Molecules
Site
Function
Cell adhesion molecule (CAM)
L Selectins
Trophinin
Endometrial pinopods,
Embryo trophoectoderm
Adhesion
Integrins
Luminal epithelium
Binds to extracellular matrix ligands
E-Cadherins
Luminal epithelium
Down regulated to facilitate trophoblast Invasion
Mucins (MUC-1)
Endometrium
Down regulated on pinopods to expose CAMs thereby selecting a good site for implantation
Cytokines
LIF
IL-1,6 & 11
CSF-1
Luminal and glandular epithelial cells
Trophoblastic growth and proliferation
Role in adhesion and invasive phase
Growth Factors
TGF-β
EGF family (EGF,TGF-β ,HB-EGF)
FGF
IGF
PDGF
Endometrium
Blastocyst (HB-EGF)
Stimulates adhesion
Increases invasiveness
Promotes decidualization
MMP (Matrix metalloproteinase)
MMP-9
TIMP-1
Endometrium & Embryo
Matrix degradation
Prostaglandins
Uterine decidualisation
Increased vascular permeability
During the proliferative phase, high oestrogen levels act via the oestrogen receptor-α (ERα) to inhibit integrin expression. The luteal progesterone rise subsequently down-regulates the number of these receptors, thus indirectly suppressing the inhibitory effects of E2 resulting in a net integrin increase. Progesterone, probably, also acts positively by increasing paracrine stromal factors, e.g. epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF) to induce epithelial ß3integrin expression that serves as the rate-limiting step in aVß3 formation.
Aberrant αVß3 integrin expression pattern has been associated with unexplained infertility [27–29], endometriosis [30], hydrosalpinx [31], luteal phase deficiency and, more recently, polycystic ovarian syndrome [32]. Hence, this integrin is a promising marker of implantation process.
Selectins are glycoproteins belonging to the cell adhesion molecule (CAM) family. The human L-selectin is of importance in the implantation process. On the blastocyst side, strong L-selectin staining has been observed over the entire embryo surface. On the maternal side, the expression of selectin oligosaccharide-based ligands, such as MECA-79 or HECA-452, is up-regulated during the window of implantation [33]. It appears that selectins take part in the very early stages of blastocyst interactions with the uterine wall.
23.4 Recurrent Implantation Failure – Endometrial Receptivity and Thickness
Recurrent implantation failure (RIF) is determined when embryos of good quality fail to implant following several in vitro fertilization (IVF) treatment cycles. A recent definition states that failure to implant in 3 IVF cycles or failure to implant after transfer of 10 good-quality embryos should be categorized as RIF. Implantation failure is related to either maternal factors or embryonic causes. Among the various potential causes of RIF, uterine factors (e.g. thin endometrium, poor endometrial receptivity and immunological incompatibility) have received the most attention in recent years. Assessing the endometrium by uterine artery blood flow indices, sub-endometrial blood flow and endometrial receptivity assay (ERAS) has been suggested (Table 23.2). More recently, endometrial biopsy in a previous cycle with electron microscopic visualization of pinopods to assess the putative implantation window and transfer of good-quality embryos during the window of implantation will improve success rates.
Table 23.2
Tests for Evaluating Endometrial Receptivity
Test | Marker | Method | Value in Implantation Failure | Relevance |
|---|---|---|---|---|
1. Endometrial (a) Molecular markers (b) Histology (c) Uterine (i) Genomics (ii) Proteomics (iii) Secretomics | αvβ3 | Flow cytometric analysis Immunohistochemistry RNA studies on Endometrial Biopsy | Decreased | Research |
LIF | Decreased | |||
E-Cadherin | Decreased | |||
MECA -79 | Absent | |||
MUC-1 | Decreased | |||
IL-10 | Decreased | |||
VEGF | Decreased | |||
eNOS | Decreased | |||
HOXA 10,11 | Decreased | |||
COX-2 | Decreased | |||
Pinopods | Scanning Electron Microscope | Poorly developed | ||
Associated genes HOX A10 Proteins IL-1β,TNF-α , IFN-ϒ, MCP-1, Glycodelin, HBEGF, VEGF | Microarray (Endometrial Biopsy) Uterine Flushing (Endometrial secretions) | Decreased | ||
2. Ultrasonography | Endometrial thickness | 2D | <7 mm | Proposed Low PPV, High NPV. Individual parameters not of sufficient accuracy to predict receptivity as compared to Uterine score |
Endometrial pattern | Not Multi layered | |||
Myometrial echogenecity | Non homogenous | |||
Endometrial volume | 3D | <2.5 ml | ||
Pulsatility index | Doppler | ≥3 | ||
Vascularisation index | ||||
Flow index | ||||
End Diastolic blood flow | Absent | |||
Protodiastolic Notch | Present | |||
3. Hysteroscopy | Synaechae Polyps Myomas | Present |
23.5 Treatment Options to Optimize Implantation
The non-hormonal adjuvant treatment of RIF ideally should be targeted to the correction of any potential malfunction that might contribute to the failure of implantation. However, since the pathological processes are poorly understood, a number of empirical treatment modalities have been tried with limited success rates. These are listed below and tabulated in Table 23.3 [34–36].
Table 23.3
Methods Used to Improve Endometrial Receptivity
Molecules | Intervention | Proposed Mechanism | Result |
|---|---|---|---|
Sildenafil | Vaginal/Oral | Increases the uterine blood flow Increases endometrial thickness | Biochemical pregnancy rates higher but no significant improvement in ongoing pregnancy rates |
Aspirin | Oral | Inhibits prostaglandin synthesis Increases uterine blood flow Reduces uterine contractions Reduces inflammation? Attenuates placental apoptosis | No evidence that use of aspirin is effective per Cochrane review 2011 [34] |
Low molecular weight heparin | Subcutaneous | Anticoagulant effect Modulates blastocyst apposition, adherence and invasion Enhances trophoblast differentiation and invasion | May benefit but avoid routine use until further research, per Cochrane Review 2013 [35] |
Granulocyte colony-stimulating factor | Subcutaneous intrauterine catheter | ? Interaction with immune system | Promising role |
Intravenous immunoglobulin | IV | Reduces NK cell activity | Lack of evidence in APA negative women |
Steroids | Oral | Immunomodulator | No clear evidence per Cochrane review 2012 [36] |
Atosiban | IV | Reduces uterine contractility? Priming of endometrium | Further studies required |
GnRH agonist in luteal phase | Subcutaenous | Improves corpus luteal function | Further studies required |
Local injury to the endometrium | Hysteroscopic procedure | Decidualisation of endometrium Production of cytokines and growth factors | Further studies required |
23.5.1 Sildenafil
Endometrial growth is thought to depend on uterine artery blood flow. Oestrogen-induced endometrial proliferation is in large part dependent upon blood flow to the basal endometrium. Nitric oxide (NO) relaxes the vascular smooth muscle by c-GMP-mediated pathway [37].
Sildenafil citrate, a type-5 phosphodiesterase inhibitor, potentiates the vasodilatory effects of NO by preventing the degradation of c-GMP [38]. Sildenafil citrate can improve the uterine blood flow and, in conjunction with oestrogen, lead to the oestrogen-induced proliferation of the endometrial lining. A good correlation has been found between endometrial thickness and the prevalence of conception. An endometrial thickness of around 9 mm on vaginal ultrasound in the late proliferative phase correlates well with the chance of pregnancy after IVF, whereas a thinner endometrium is associated with poorer implantation rates [39].
Sildenafil citrate improves the uterine artery blood flow and the sonographic endometrial thickening in patients with a poor outcome in a prior assisted reproductive treatment (ART) cycle due to poor endometrial response [40, 41]. The biochemical pregnancy rates are also higher with sildenafil citrate but do not reach statistical significance [41].
Although NO improves uterine blood flow in the proliferative phase, it may have detrimental effects on the endometrium during the implantation window. The NO-mediated release of cytokines like TNFα from the activated natural killer cells has been implicated as a cause of implantation failure [42]. Hence, it may be beneficial to minimize endometrial exposure to NO at the time of embryo transfer by discontinuing sildenafil on or prior to the day of HCG administration.
Nitroglycerine (NTG) patch also improves the endometrial blood flow and lining in IVF patients with a previous poor response but is associated with side effects like headaches and hypotension. The use of sildenafil vaginal suppositories (25 mg) decreases systemic side effects and is preferred over NTG patches.
23.5.2 Aspirin
Low-dose acetylsalicylic acid (aspirin) irreversibly inhibits the cyclo-oxygenase enzyme in platelets, thus preventing the synthesis of thromboxane, which causes vasoconstriction and platelet aggregation [43]. By this mechanism, low-dose aspirin may enhance uterine blood flow and tissue perfusion, thereby improving endometrial receptivity for implantation. Aspirin may also suppress negative effects of prostaglandins on implantation, such as the induction of uterine contractions or inflammatory response.
In vitro studies have shown that heparin and aspirin attenuate placental apoptosis, and this could be a possible explanation of how aspirin is beneficial, even in the absence of endometrial or oocyte improvement [44]. This theory along with its low cost, free availability and minimal side effects has popularized the use of low-dose aspirin in ART cycles.
Several studies have shown that aspirin is beneficial in infertility [45]. A non-controlled study found that IVF outcome was significantly improved when aspirin, heparin and intravenous immunoglobulin therapy was administered to women with repeat IVF failures and anti-phospholipid antibodies but not to women with negative anti-phospholipid antibodies [46, 47].
However, Cochrane review of 2011 concluded that there is no evidence that the use of aspirin in women undergoing IVF is effective [34]. A study on the effect of aspirin in uterine haemodynamics among unselected IVF/ICSI women revealed that low-dose aspirin therapy 100 mg/day, when started concomitantly with gonadotropin stimulation, does not significantly affect uterine artery vascular impedance or endometrial thickness on the day of embryo transfer [48].
A recent meta-analysis concluded that use of aspirin does not improve success rates in IVF cycles [49].
23.5.3 Low Molecular Weight Heparin (LMWH)
Many studies have reported congenital and acquired coagulation defects to be more prevalent in women with recurrent implantation failures (RIFs) [50]. This led to the use of anti-coagulants, mainly heparin, during the course of ART cycles in women with anti-phospholipid antibodies [50–52].
Heparin is a linear polydisperse polysaccharide consisting of 1 → 4-linked pyranosyluronic acid and 2-amino-deoxyglucopyranose (glucosamine) residues [53]. Due to the highly anionic nature, heparin can bind to a plethora of proteins including anti-thrombin, growth factors, growth factor receptors, viral envelope proteins and extracellular matrix molecules.
The changes in coagulation and fibrinolysis observed during ovarian stimulation are similar to those observed during pregnancy, with the drive for these haemostatic changes potentially being the rapid increase of oestradiol levels, which occur with ovarian stimulation [54]. Excessive coagulation activation was found to be associated with poorer IVF outcomes, despite higher oocyte yields. This suggests that haemostatic mechanisms have an important role in implantation. Heparin can alter the haemostatic response to controlled ovarian stimulation and modify the risk of thrombosis.
Heparin has been proposed to play a role in the process of implantation beyond its anti-coagulant effects, through interactions with several adhesion molecules, growth factors, cytokines and enzymes such as matrix metalloproteinases (MMP). It can also modulate many of the fundamental physiological processes required for blastocyst apposition, adherence and invasion. It enhances trophoblast differentiation and invasion and has the potential to improve pregnancy rates and outcomes in ART cycles [53].
E-cadherin expression by the endometrium is decreased by progesterone facilitating trophoblast invasion. Unfractionated heparin (UFH) and enoxaparin, a LMWH, have also been shown to down-regulate decidual E-cadherin expression [55], thereby potentially explaining the observations that UFH and LMWH can promote extra-villous trophoblast differentiation [56].
HB-EGF is induced by sex steroids during the secretory phase of the endometrial cycle and persists during early pregnancy [57]. Its expression on the surface of pinopods [58] suggests an early role in blastocyst implantation and placentation. LMWH may potentiate sHB-EGF binding and may also up-regulate sHB-EGF levels via increased MMP activity.
Interleukin -1 (IL-1) increases endometrial epithelial cell β3 integrin expression with an improvement in blastocyst adhesion [59]. LMWH is reported to increase IL-1 expression in activated leukocytes [60]. Modulation of integrin expression by LMWH may be playing a role in improving endometrial receptivity. Enhanced trophoblast migration and invasiveness due to LMWH-induced increase in free insulin-like growth factor I is another proposed mechanism for a beneficial effect of LMWH on the implantation process.
A pilot study on luteal phase empirical LMWH (1 mg/kg/day) a day after oocyte retrieval in RIF patients observed a relative increase by 30 % in live birth rates. Though the difference was not statistically significant, it suggested a potential beneficial effect of LMWH on the clinical outcome of ART in women with RIF. UFH as well as LMWHs are able to modulate the decidualization of human endometrial stromal cells in vitro and therefore might be useful to control endometrial differentiation and receptivity in assisted reproduction [61].
A recent prospective randomized study observed significant differences with regard to pregnancy and implantation rates in ICSI patients treated with combined oral prednisolone and LMWH in unexplained failed implantation [62].
The results of a Cochrane review of three randomized controlled trials with a total of 386 women suggested that peri-implantation LMWH in ART cycles may improve the live birth rate. However, the results were dependent on small low-quality studies with substantial heterogeneity and were sensitive to the choice of statistical model. There are side effects reported with use of heparin, including osteopenia, bruising and bleeding, with no reliable data on long-term effects. Currently, the use of heparin outside well-conducted research trials is not justified [35]. Patients in whom LMWH would be most effective and the appropriate dosing and duration of administration needs to be determined before unselectively exposing women and their embryos to this medication.
23.5.4 Granulocyte Colony-Stimulating Factor
G-CSF is a cytokine with a 177 amino acid polypeptide chain and a molecular weight of 25 kDa. It stimulates neutrophilic granulocyte proliferation and differentiation. It is expressed and produced by the decidual cells, and its receptor, c–fms, is expressed on the trophoblastic cells [63].
Scarpellini et al. in 2009 studied the efficacy and safety of G-CSF in women with unexplained recurrent miscarriage with at least four consecutive miscarriages and negative for all clinical investigations. Recombinant G-CSF was administered subcutaneously daily at a dosage of 1 mg (100,000 IU)/kg/day from the sixth day after ovulation until the occurrence of menstruation or to the end of the ninth week of gestation. The number of live births in women treated with G-CSF was significantly higher as compared to controls. Also, elevated levels of beta-hCG were observed during treatment with G-CSF showing thereby that G-CSF may increase the trophoblast growth and metabolism. The side effects included skin rash and leucocyte count higher than 25,000/ml. None of the newborns showed any major or minor abnormalities or malformations [64].
Presence of chronically thin endometrium, resistant to standard treatments, affects a small number of patients undergoing IVF. Endometrial thickness below 7 mm is widely considered sub-optimal for transfer and associated with reduced pregnancy chances [65].
Gleicher et al. in 2012 reported the successful use of G-CSF in those who had previously failed to expand their endometria beyond 6.9 mm with the use of standard treatments. Infertile women with endometrial thickness of <7 mm on the day of hCG administration in their first IVF cycles and in whom traditional treatments with oestradiol, sildenafil citrate and beta blockers had been unsuccessful were administered G-CSF by intra-uterine catheter by slow infusion before noon on the day of hCG administration. If the endometrium had not reached at least 7-mm within 48 h, a second infusion was given following oocyte retrieval. A significant improvement in endometrial thickness after G-CSF treatment was reported [66].
Even though there is increasing evidence that G-CSF is not toxic in pregnancy, it should be used very carefully as its safety is still under question and there are not enough women treated with G-CSF in pregnancy to exclude any possible teratogenic effects. There is still little knowledge of the role of G-CSF in human reproduction and its interaction with the immune system, but it has a promising role in those with refractory thin endometrium.
23.5.5 Intravenous Immunoglobulin (IVIg)
Women experiencing implantation failure have a higher frequency of elevated percentage of circulating CD56+ (natural killer) cells (>12 %) than fertile women (3–12 %). IVIg reduces activation of NK cells and NK killing activity both in vitro and in vivo. IVIg in doses of 500 mg/kg prior to embryo transfer significantly improved the pregnancy rates in women with elevated percentage of circulating CD56+ cells [67].
IVIg may be a useful treatment option for patients with previous IVF failure and preconception Th1:Th2 imbalance and/or NK elevation. Preconception immune testing may be a critical tool for determining which patients will benefit from IVIG therapy [68, 69]. IVF outcome was reported to be significantly improved when heparin/aspirin and IVIG was administered to anti-phospholipid antibody (APA)-positive women with repeat IVF failures whereas APA negative women did not seem to benefit from such treatment [47].
IVIg treatment for repeated IVF/ICSI failure and unexplained infertility was reported to significantly increase implantation and pregnancy rates in a systematic review and meta-analysis [70].
Recently, a systematic review of literature on interventions to improve reproductive outcomes in women with elevated natural killer cells undergoing ART does not support the use of prednisolone, IVIg or any other adjuvant treatment in women undergoing ART who are found to have elevated absolute numbers or activity of NK cells, due to the paucity of or poor quality of the evidence [71]. Further research is needed before NK cell assessment can be recommended as a diagnostic tool in the context of female infertility or recurrent miscarriage.
23.5.6 Steroids
It has been proposed that glucocorticoids may improve the intra-uterine environment by acting as immune modulators to reduce the uterine NK cell count, normalize cytokine expression profile in the endometrium and suppress subclinical endometrial inflammation.
Several studies have reported that immunosuppressive doses of corticosteroids administered for a short period of time to patients undergoing IVF-ET can significantly improve the implantation and pregnancy rates [72], especially in those with associated autoimmune conditions [73]. A study reported that prednisolone reduces pre-conceptual endometrial NK cells in women with recurrent miscarriage [74]. However, some studies have shown no improvement in implantation and pregnancy rates in glucocorticoid-treated patients [75].
The Cochrane review (2012) concluded that there is no clear evidence that administration of peri-implantation glucocorticoids in ART cycles significantly improves the clinical outcome. The use of glucocorticoids in a sub-group of women undergoing IVF (rather than ICSI) was associated with an improvement in pregnancy rates of borderline statistical significance and should be interpreted with care. These findings were limited to the routine use of glucocorticoids and cannot be extrapolated to women with autoantibodies, unexplained infertility or recurrent implantation failure [36]. Further well-designed randomized studies are required to elucidate the possible role of this therapy in well-defined patient groups.
23.5.7 Atosiban
Uterine contractile activity may adversely affect implantation. Increased contractions have been found in approximately 30 % of patients undergoing embryo transfer. Pharmacological tocolytics may be expected to improve pregnancy rates; however, targeting uterine adrenergic receptors, calcium channels or prostaglandin synthesis has been ineffective.
Oxytocin antagonist atosiban is being used as a tocolytic to delay premature labour by inhibiting contractions of the uterus. Atosiban given at the time of embryo transfer to women with recurrent implantation failure reduced the number of uterine contractions in these patients and also increased the implantation and pregnancy rates. The pregnancy rate went from zero to 43.7 %. The total dose of atosiban was 36.75 mg [76].
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