Repeated Embryo Implantation Failure (RIF)

Abstract: On the Possible Origins of the Repeated Embryo Implantation Failure. Causes. Consequences. Dilemmas. Breakthroughs. Perspectives. 

The article focuses on researching the peculiarities of the whole process of the Embryo Implantation and on discussing the possible origins of the repeated Embryo Implantation failure. Special emphasis is given to the basic overview of the causes and further consequences of the Recurrent Implantation Failure. As a result of integrative studying of the causes and further consequences, it should be noted that many dilemmas of the hypothesis, concepts and theories considering this issue must be further discussed.

Introduction.

Pregnancy is a unique phenomenon, which presupposes synchronic integrative interaction of many mechanisms. To occur, a competent embryo must attach to a receptive endometrial lining and then invade the underlying decidualizing stroma. Although the scientific knowledge of the molecular mechanisms that govern these early embryo–maternal interactions has increased substantially in recent years, Embryo Implantation remains the least understood phenomenon in Assisted Reproductive Technology (ART). The delicate and comprehensive process of Embryo Implantation, a remarkably dynamic and precisely controlled molecular and cellular event, sometimes appears inefficient. Insights gained from clinical implantation failure, early pregnancy loss, and emerging technologies that enable molecular interrogation of endometrial–embryo interactions have led the experts to the conclusion, that the process of Embryo Implantation and all its mechanisms should be investigated inclusively through numerous concepts and theories’ paradigms.

Similarly, it would be difficult to determine inclusively and conclusively the most accurate theory which would transparently represent the whole paradigm of indicators, factors and causes which lead to Recurrent Implantation Failure (RIF) or early embryonic mortality and would represent the most effective preventive measures to avoid the negative consequences for the embryo development. Consequently, formulating the structure of the above–mentioned paradigm presupposes: 

1. Substantiating the accurate understanding of the first indicators and mechanisms of Embryo Implantation failure based on clinical evidence and emerging concepts would reveal the association between embryo and maternal interactions which reflect both biological mechanisms and forces of Embryo Selection; 
2. Analyzing the peculiarities of Embryo Implantation process and identifying the underlying mechanisms of implantation failure; 
3. Formulating the clinical prognosis for Embryo Implantation process; 
4. Implementing the preventive measures for Embryo Implantation failure or repeated implantation failure in clinical practice.

Broadly defined, pregnancy loss includes any type of loss of the conceptus from fertilized oocyte to neonate [ACOG, 2002]. This definition, given by ACOG, is inclusively interpreted as ‘repetitive loss of recognized pregnancies in the first or early second trimester (<15 weeks of gestation)’ [ACOG, 2002]. It usually is referred to as recurrent spontaneous abortion, miscarriage, or recurrent early pregnancy loss [ACOG, 2002]. Thus, repeated Embryo Implantation failure or recurrent pregnancy loss is a complicated problem facing many couples, researchers, doctors and clinicians.

Despite tremendous breakthroughs and continuing advances in Assisted Reproductive Technology (ART), it is well agreed upon that implantation is the success–limiting event in Assisted Reproductive Technique (ART) and possibly the major contributory factor restricting progress in Assisted Reproductive Technology [Koot et al., 2012; Chicea et al., 2013]. Consequently, it should be noted that all In Vitro Fertilization (IVF) programmes include patients who fail to become pregnant despite numerous Embryo Transfer procedures. Although embryo quality in Assisted Reproductive Technology (ART) has been optimized to a considerable extent, improving the receptive status of the endometrium significantly remains a challenging task [von Grothusen et al., 2014].

Early embryonic mortality is very high and it has been postulated that the largest single cause of failed pregnancy is an error of Embryo Implantation [Bulleti et al., 1996; Bischof et al., 2006; Christiansen et al., 2006]. Considering the fact that early embryonic mortality is highly debated theme with many controversial hypotheses, it is essential to present its average statistics, which is based on verification of clinical data: spontaneous miscarriage may be as high as 60–80% if one takes into consideration those miscarriages occurring within the first months of conception, which usually go undetected by patients [Bulleti et al., 1996; Choudhury and Knapp, 2000]. It is absolutely incontrovertible fact that the miscarriages affect nearly 15% of women, primarily in the first trimester, and while most are sporadic and non–recurrent, there is a subset comprising 2–5% of couples that suffers Recurrent Implantation Failure (RIF) or, in other words, Recurrent Miscarriage (RM) [Clark et al., 2001]. These recurrent embryo losses suggest the presence of a specific cause, and much work should be carried out by the clinicians and researchers to try to identify the underlying mechanisms.

Recurrent Implantation Failure (RIF) following Embryo Transfer (ET) is a major undissolved and continuing problem in In–Vitro Fertilization (IVF) Treatment therapy. Recurrent Implantation Failure (RIF) or Recurrent Miscarriages (RM) during In Vitro Fertilization–Embryo Transfer (IVF–ET) refers to a clinical condition where implantation has failed repeatedly, despite several successful transfers of good quality embryos [Simon and Laufer, 2012a,b; Roy Choudhury et al., 2016]. Despite Recurrent Implantation Failure (RIF) remaining a controversial and highly–debated topic amongst researchers and clinicians, there are no formal criteria defining the total number of embryos to be transferred or the number of failed cycles to occur during In Vitro Fertilization (IVF) therapy [Angeles Martínez–Zamora M., 2011].

It was postulated that Recurrent Implantation Failure (RIF) or Recurrent Miscarriage (RM) is a heterogeneous condition. A large number of current studies has recently been published, yet many of them have conflicting conclusions with the special emphasis on suggestion that the direct causes are identified in no more than 50% of couples, and several alleged causes of recurrent pregnancy loss are controversial [ACOG, 2002; Li et al., 2002]. Remarkably, as recently stressed, it is possible that Recurrent Miscarriage (RM) and Recurrent Implantation Failure (RIF) may, in some situations, represent different manifestations of the same pathogenic spectrum [Angeles Martínez–Zamora M., 2011] and in fact it has been documented that some abnormalities are associated with both implantation failure and miscarriage [Christiansen et al., 2006; Stern and Chamley, 2006].

Basically, there are two vital indicators of the successful Embryo Implantation: well–functioning endometrium and healthy embryo. An abnormality in one or both these variables can cause Repeat Implantation Failure (RIF). 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 Recurrent Implantation Failure (RIF) [Tan et al., 2005]. Today with the tendency of transferring only one or two embryos, the definition of Recurrent Implantation Failure (RIF) is not apparent, because there are no strict inclusion/exclusion criteria [Margalioth et al., 2006]. Nevertheless, Margalioth et al. (2006) suggest that after failure of three cycles in which reasonably good embryos were transferred, further investigation should be initiated [Margalioth et al., 2006]. Implantation failure is related to either maternal factors, embryonic defects or multifactorial causes. Maternal factors include uterine anatomic abnormalities, thrombophilia, non–receptive endometrium and immunological factors. Failure of implantation due to embryonic causes is associated with either genetic abnormalities or other factors intrinsic to the embryo that impair its ability to develop in utero, to hatch and to implant [Simon and Laufer, 2012]. In other words, reduced endometrial receptivity and, sometimes, poor embryo quality and inappropriate transfer techniques are considered to be the major causes of Recurrent Implantation Failure (RIF) [Margalioth et al., 2006]. An excellent review by Coughlan et al. (2014) discusses Recurrent Implantation Failure (RIF) in great detail [Coughlan et al., 2014].

Additionally, what should be taken into consideration, is that Recurrent Implantation Failure (RIF) could be caused by many different factors including the various integrative interactions of these factors, such as inappropriate Ovarian Stimulation, suboptimal laboratory culture conditions and faults in Embryo Transfer techniques [Margalioth et al., 2006].

1. Embryo Implantation and implantation failure: molecular dimension. Human Chorionic Gonadotrophin (hCG) as the extremely essential indicator of the prevalence of implantation events after In Vitro Fertilization: the concepts and conclusions represented in the study “Molecular aspects of implantation failure” written by Koot Y.E., Teklenburg G., Salker M.S., Brosens J.J., Macklon N.S.

Implantation is considered to occur when a blastocyst breaches the luminal endometrial epithelium. However, determining precisely when this occurs is very complicated. The only established clinical marker of implantation is human Chorionic Gonadotrophin (hCG) [Koot et al., 2012]. This glycoprotein hormone, produced by the cytotrophoblast cells of the blastocyst, is first detectable in urine and blood a few days after implantation and then rises exponentially [Marshall et al., 1968; Armstrong et al., 1984; Canfield et al., 1987]. In Assisted Reproductive Technology (ART), implantation is defined as a quantitative rise in human Chorionic Gonadotrophin (hCG) level above a threshold level at some point after Embryo Transfer [Rinehart, 2007]. 

The most accurate way to determine the prevalence of implantation events after In Vitro Fertilization (IVF) would be to measure human Chorionic Gonadotrophin (hCG) levels in a daily serum sample after Embryo Transfer [Koot et al., 2012].

The earlier human Chorionic Gonadotrophin (hCG) is measured, the more implantation events will be captured, including transient events [Koot et al., 2012]. Consequently, more cases of early post–implantation pregnancy loss will also be detected [Wilcox et al., 1988; Wang et al., 2003; Boomsma et al., 2009]. Thus, the frequency of implantation and implantation failure depends on detection methods and clinical definition [Koot et al., 2012].

The clinical definition of ‘Recurrent’ Implantation Failure (RIF) is very challenging. The 2005 ESHRE Preimplantation Genetic Diagnosis (PGD) consortium defined the criteria for Recurrent Implantation Failure (RIF) as the absence of implantation after ≥ 3 Embryo Transfers with high–quality embryos or after replacement of a total of 10 or more embryos in multiple transfers, with the special emphasis on the exact numbers to be determined by each Fertility Clinic [Thornhill et al., 2005]. Therefore, a definition of Recurrent Implantation Failure (RIF) as three failed In Vitro Fertilization (IVF) cycles is inevitably arbitrary. Further, the definition should include the number of good–quality embryos transferred without achieving a pregnancy. This inclusion criterion is increasingly applied in recent studies [Blockeel et al., 2008; Koler et al., 2009; Ledee et al., 2011; Léddée et al., 2011].

2. Overview of the assumed aetiologies for embryo Recurrent Implantation Failure (RIF): the concepts and conclusions represented in the study “Investigation and Treatment of repeated implantation failure following IVF–ET” written by Margalioth E.J., Ben–Chetrit A., Gal M., Eldar–Geva T. S., Brosens J.J., Macklon N.S.

Embryonic loss, which occurs repeatedly after Assisted Reproduction, may be attributed to many different factors. 

These factors can be presented into three categories: 

1. Decreased endometrial receptivity; 
2. Embryonic defects and 
3. Factors with combined effect [Margalioth et al., 2006].

1). Decreased endometrial receptivity: 

1. Uterine cavity abnormalities; 
2. Thin endometrium; 
3. Altered expression of adhesive molecules; 
4. Immunological factors; 
5. Thrombophilias.

2). Defective embryonic development: 

1. Genetic abnormalities (male/female/gametes/embryos); 
2. Zona hardening; 
3. Suboptimal culture conditions.

3). Multifactorial effectors: 

1. Endometriosis; 
2. Hydrosalpinges; 
3. Suboptimal ovarian stimulation

Short overview of the factors, included into “Decreased endometrial receptivity” category

Whilst it is well known that much of the failure of Embryo Implantation may be explained by chromosomal abnormalities, it has been postulated that in some patients there may be a problem with uterine receptivity. This, hypothetically, could be at the level of an unfavourable intra‐uterine milieu or an endometrial lining that is hostile to embryonic implantation. Recurrent Implantation Failure (RIF) might be caused by undiagnosed uterine pathology or uterine abnormalities, mainly hyperplasia, polyps, endometritis, synechiae and leiomyomata [Demirol and Gurgan, 2004]. In natural pregnancies, the developing embryo usually enters the uterine cavity at the morula stage and spends 2–3 days within the cavity prior to implantation at the blastocyst stage. Successful implantation therefore depends upon appropriate intra–uterine conditions as well as a receptive endometrium. In the case of Embryo Transfer following In Vitro Fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI), embryos are commonly transferred into the uterine cavity at day 2 or 3, and spend 4 or 3 days respectively in the uterine cavity prior to implantation. The difficulties encountered by many IVF units in establishing efficient extended in–vitro blastocyst culture systems reinforces the importance of the extra–embryonic environment during this pre–implantation phase [Inagaki et al., 2003].

The presence of a thin endometrium 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 Intracytoplasmic Sperm Injection (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 Recurrent Implantation Failure (RIF) cases [Margalioth et al., 2006].

Some cases of Recurrent Implantation Failure (RIF) are related to dysregulation of the normal expression or action of various cytokines. Elevated endometrial NK cells, dysregulation of Interleukin (IL) 12, 15 and 18 [Ledee–Bataille et al., 2005], high IL–1β and low interferon–γ and IL–10 [Inagaki et al., 2003] can be found in Recurrent Implantation Failure (RIF). Failure of appearance of a specific 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 matrix metalloproteinases [Inagaki et al., 2003] have been suggested to be closely associated with Recurrent Implantation Failure (RIF) of the embryo.

The role of immunological causes and thrombophilia in implantation failure through mechanisms similar to recurrent miscarriages has been the focus of many recent research efforts, because the reproductive failure may be a consequence of aberrant expression of immunological factors during pregnancy. Thorough analysis and investigation which were done by Shormila Roy Choudhury and Leslie A. Knapp vividly illustrated the following paradigm: although the relative importance of immunological factors in reproduction remains controversial, substantial evidence suggests that Leukocyte Antigens (HLA), antisperm antibodies, integrins, the Leukaemia Inhibitory Factor (LIF), cytokines, antiphospholipid antibodies, endometrial adhesion factors, Mucins (MUC1) and uterine natural killer cells contribute to reproductive failure. In contrast, fewer data support the roles of anti–trophoblast antibodies, anti–endometrial antibodies, T–cells, peripheral natural killer cells, anti–HLA antibodies, blocking antibodies and suppressor cells in reproductive failure [Roy Choudhury and Knapp, 2001]. Although immunological factors involved in reproductive failure have been studied traditionally using assays for antibodies and/or antigens, detailed research on these factors demonstrates conflicting results. Maternal and fetal immunology is also difficult to investigate. For these reasons, molecular assays may serve as a valuable alternative to investigate how the immune system affects reproductive outcome. Ultimately, Margalioth et al. (2006) established that the significantly decreased expression of specific endometrial molecules suggested that functional, not only morphological, endometrial defects may be associated with unexplained infertility or Recurrent Implantation Failure (RIF) [Margalioth et al., 2006].

Short overview of the factors, included into “Defective embryonic development” category

It is considered that poor embryo quality is the major cause of implantation failure [Urman et al., 2005], and by and large reflects the high incidence of chromosomal abnormalities reported for human embryos [Munne 2001; Delhanty, 2005; Vanneste et al., 2009]. These abnormalities may arise from an error during meiosis, resulting in a uniform abnormality present in all cells, or from segregation errors occurring during the first mitotic divisions, resulting in chromosomal mosaicism [Koot et al., 2012], for instance, the scientific group under the management of Vanneste E. [Vanneste et al., 2009], revealed not only mosaicism for whole–chromosome aneuploidies and uniparental disomies in most cleavage–stage embryos but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage–fusion–bridge cycles, what explains the low fecundity and identifies post–zygotic chromosome instability as a leading cause of constitutional chromosomal disorders [Vanneste et al., 2009]. Mosaicism has been reported to affect up to 91% of embryos in the early stages of pre–implantation development [Baart et al., 2006; Mantzouratou et al., 2007; Santos et al., 2010]. However, the incidence of mosaicism is significantly lower when the embryo reaches the blastocyst stage [Santos et al., 2010], which could be explained by the developmental arrest of a significant proportion of mosaic day 4 embryos and/or reduced proliferation or selective apoptosis of aneuploid blastomeres within a mosaic embryo [Santos et al., 2010]. At the blastocyst stage, a majority of embryos are thought to be uniformly euploid [Northrop et al., 2010]. Chromosomal abnormalities of the male or female partner, the gametes or the developing embryo may burden embryogenesis. Increased frequency of female chromosomal abnormalities such as translocations, mosaics, inversion, deletion and chromosomal breakages, particularly at the centromere region were observed in young women with high–order Recurrent Implantation Failure (RIF) [Tarlatzis et al., 2000; Raziel et al., 2002]. 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 embryos, caused by maternal cytoplasmic factors or mutations in cell cycle control genes, might be a common cause for Recurrent Implantation Failure (RIF). Thus, it can be assumed that many patients with Recurrent Implantation Failure (RIF) develop a high percentage of chromosomally abnormal embryos that fail to implant despite good morphology and developmental rate.

Prior to fertilization, the oocyte’s surrounding zona pellucida acts as a specific sperm barrier and is involved in sperm binding. The zona pellucida (which surrounds the oocyte), hardens naturally after fertilization to prevent polyspermic fertilization and to protect the integrity of the preimplantation embryo during early embryonic development. Increased zona thickness was 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]. Thus, failure of the zona to rupture has been suggested as a possible cause of Recurrent Implantation Failure (RIF).

In some cases, Recurrent Implantation Failure (RIF) may be interconnected to an inability of the embryo to hatch out of its zona pellucida [Cohen et al., 1990]. The zona pellucida is a glycoprotein layer, surrounding the oocyte or embryo which after fertilization, compresses and shapes the embryo. The zona facilitates the active transport of the embryo trough the Fallopian tubes and protects it from micro–organisms and immune cells [Zhao and Dean, 2002]. At the blastocyst stage, the embryo needs to break out of the zona to enable invasion of the luminal endometrial epithelium. Failure of this process could be caused by zona hardening arising from IVF culture conditions or Cryopreservation. Further, advanced endometrial development caused by ovarian stimulation [Horcajadas et al., 2008] combined with delayed development of embryos in vitro [Hsu et al., 1999] could cause a synchronization problem, which requires the embryo to hatch out of the zona prematurely.

1.3. Short overview of the factors, included into “Multifactorial effectors” category

Endometriosis as a cause for Recurrent Implantation Failure (RIF) has not been investigated directly; however, all markers of reproductive process, including ovarian response, embryo quality, implantation and pregnancy rates, are decreased in endometriosis, especially in severe disease. the effect of endometriosis is not exclusively on the receptivity of the endometrium but also on the development of the oocyte and embryo [Barnhart et al., 2002].

Women with hydrosalpinges have lower implantation and pregnancy rates [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.

Endometrial and embryo qualities may be harmed by certain drugs given for ovarian stimulation. Recent Randomized Controlled Trials (RCTs) found no evidence of clinical superiority for rFSH over urinary–FSH/hMG [Al–Inany et al., 2003], for recombinant–hCG over urinary–hCG [Al–Inany et al., 2005] or for FSH/GnRH–antagonist over FSH/GnRH–agonist protocols [Barmat et al., 2005]. However, the importance of different drugs in RIF is unknown.

Conclusion

The relatively low rate of successful implantation following Embryo Transfer after In Vitro Fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI) can be partly explained by intrinsic problems within the embryos transferred. Despite tremendous breakthroughs and continuing advances in Assisted Reproductive Technology (ART), it is well agreed upon that implantation is the success–limiting event in Assisted Reproductive Technique (ART) and possibly the major contributory factor restricting progress in assisted reproductive technology [Koot et al., 2012; Chicea et al., 2013]. Consequently, it should be noted that all In Vitro Fertilization (IVF) programmes include patients who fail to become pregnant despite numerous Embryo Transfer procedures. Implantation failure could be caused by many different factors such as inappropriate ovarian stimulation, suboptimal laboratory culture conditions and faults in Embryo Transfer techniques.

Repeated Implantation Failure (RIF) is defined as failure to conceive following two or three Embryo Transfer cycles, or cumulative transfer of >10 good quality embryos [El–Toukhy and Taranissi, 2006] or is defined as failure of embryos to implant in at least three consecutive In Vitro Fertilization (IVF) Treatment cycles, where multiple high–grade embryos are transferred per cycle [Simon and Laufer, 2012a,b; Ghosh et al., 2013]. Implantation failure is related to either maternal factors or embryonic causes. Maternal factors include uterine anatomic abnormalities, thrombophilia, non–receptive endometrium and immunological factors [Salim et al., 2002].

From the clinical perspective, in all cases of Recurrent Implantation Failure or after three failures, the scientists highly recommend repeated hysteroscopy and a try of blastocyst transfer. For the effective preventing of Recurrent Implantation Failure, the main emphasis should be given to four constituents of the inclusive paradigm in every clinical case: 

1. Substantiation of the accurate understanding of the first indicators and mechanisms of Embryo Implantation failure based on clinical evidence through investigation the association between embryo and maternal interactions; 
2. Analysis of the peculiarities of Embryo Implantation process and identification of the underlying mechanisms of implantation failure; 
3. Formulation of the clinical prognosis for Embryo Implantation process; 
4. Implementation of the preventive measures for Embryo Implantation failure in the next IVF treatment therapy’s cycle.

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