Principles and Practice of Controlled Ovarian Stimulation in ART 1st ed.

23. Endometrial Support Beyond Hormones

Mala Arora  and Shilpa Gulati2

(1)

Infertility and IVF, Noble IVF Centre, Sector-14, Market, Faridabad, Haryana, 121007, India

(2)

Department of Obstetrics and Gynaecology, Institute of Reproductive Medicine, Kolkata, West Bengal, India

Mala Arora

Email: narindamala@gmail.com

Abstract

Implantation is a very complex process, which is controlled by a number of molecules like hormones, cytokines and growth factors and their crosstalk. During the implantation period, the endometrium acquires an appropriate morphological and functional state under the influence of ovarian steroids and molecular mediators. Assisted reproductive technology protocols continue to evolve with the aim of achieving higher pregnancy rates; however, despite these advances, implantation rates are still relatively low and have not increased sufficiently in the last decade to allow widespread adoption of single-embryo transfer.

A number of empirical treatment modalities have been tried but with limited success rates, since the pathological processes are poorly understood. Endometrial stem cells and gene therapy are promising options that can be effective in the future. Use of new tissue profiling technologies at genomic, transcriptomic and proteomic levels will bring new strategies in treating implantation failure and help increase successful pregnancies. This chapter aims to summarize the current knowledge of the mechanism of implantation, molecular and morphological markers of endometrial receptivity and proposed treatment options to improve implantation rate.

Keywords

EndometriumReceptivityImplantationGranulocyte colony stimulating factorAspirinHeparinSildinafil

23.1 Introduction

Embryo implantation is the most critical step of the reproductive process. It consists of a unique biological phenomenon, by which the blastocyst becomes intimately connected to the maternal endometrial surface to form the placenta that will provide an interface between the growing foetus and the maternal circulation [1].

The process of implantation is subdivided into the stages of

·               Apposition

·               Adhesion

·               Invasion

Prior to implantation, the blastocyst shows evidence of polarity, assuming a particular orientation as it approaches the endometrium. Once the blastocyst is oriented correctly (apposition), the zona pellucida is shed. The blastocyst then comes into contact with the endometrial surface and adheres to an endometrial gland opening, drawing nutrition from its secretions (adhesion). Finally, the blastocyst penetrates the surface layer and invades the stroma (invasion) [2].

Successful implantation requires the appropriately timed arrival of a viable blastocyst into a receptive endometrium and a synchronized dialogue between maternal and embryonic tissues [3]. The endometrium is remodelled throughout the menstrual cycle and exhibits only a short period of receptivity, known as the ‘implantation window’. In humans, during a natural cycle, the embryo enters the uterine cavity 4 days after ovulation [4]. The endometrium becomes receptive to blastocyst implantation 6–8 days after ovulation and remains receptive for approximately 4 days (cycle days 20–24) [5].

Implantation failure remains an unsolved problem in reproductive medicine and is considered as a major cause of unexplained infertility in otherwise healthy couples. Indeed, the average implantation rate in IVF is around 25 % [6]. Inadequate uterine receptivity is responsible for approximately two-thirds of implantation failures, whereas the embryo itself is responsible for only one-third of these failures [7].

Hence, to improve implantation rates in stimulated cycles, it is important to pinpoint the window of implantation, ensure that the best embryo is selected and synchronize embryo transfer with the time of optimal endometrial receptivity. There is a need to identify ways of evaluating and enhancing endometrial receptivity and embryo quality to maximize implantation rates in ART cycles.

Throughout the menstrual cycle, the human endometrium is primed for blastocyst attachment. Hence, it needs to acquire an accurate morphological and functional state. A large number of molecular mediators, under the influence of ovarian hormones, have been postulated to be involved in this early foeto–maternal interaction. These mediators include a large variety of inter-related molecules including adhesion molecules, cytokines, growth factors, lipids and others [8].

23.2 Immunology of Successful Pregnancy

Immune responses play an important role in embryo implantation. Medawar in 1953 gave the concept that the foetus represents a semi-allograft developing in the potentially hostile environment of the maternal immune system. Immune responses play a very important role, so that the mother accepts a semi-allogeneic foetus [910], The main tissue where maternal allo-recognition of the foetus occurs is in the uterus at the site of placentation, where fetal extra-villous trophoblast cells invade and intermingle with maternal leukocytes. About 40 % of recurrent miscarriages are unexplained and immune dysfunction or allo-immune responses may be responsible.

A number of cytokines and their receptors are expressed at the materno-foetal interface and are thought to play a function in the regulation of placentation. In pregnancy, there is a shift from Th type 1 cytokine production to Th type 2, since type 1 cytokines (e.g. IFN-g and TNF-a) are harmful for pregnancy as they may inhibit successful implantation [1112].

Unexplained recurrent miscarriages could be due to an imbalance between Th1/Th2 systems. If there is increased production of cytotoxic Th1 cytokines (interleukin 2, TNFα), instead of Th2 cytokines (interleukin 4, 6 and 10) which have an immunosuppressant role, it will result in rejection of embryonic allograft. [13] Uterine NK cells account for approximately 70 % of decidual leukocytes and are likely to be involved in the process of placentation. They increase markedly in early pregnancy. To escape lysis by uNK cells, the trophoblast cells express the MHC Ib antigens, HLA-E and HLA-G. Inhibitory KillerIg-like receptors (KIRs) interact with foetal HLA-C in the early weeks of gestation and prevent lysis of the trophoblast cells [14].

The trophoblast invades the decidua to surround and destroy the media of the spiral arteries, transforming them into high-conductance vessels. A role for uNK cells in implantation and placentation is suggested by the findings that high pre-conceptional NK activity was associated with significantly higher rates of miscarriage [15] and infertility [16]. The uNK cell-derived cytokines influence placentation. Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and leukaemia inhibitory factor (LIF) stimulate growth of the trophoblast; colony-stimulating factors also promote trophoblast cell proliferation and differentiation [1718].

23.3 Assessment of Endometrial Receptivity (Morphological and Molecular Markers)

The endometrium is a multilayered, dynamic organ comprising of a functional layer and a basal layer. The cells in the functional layer are shed during menstruation. The basal layer is attached to the myometrium and remains intact during menstruation, serving as a base for endometrial regeneration. The endometrium is composed of several different cell types, including luminal and glandular epithelial cells, stroma with stromal fibroblastic cells, immunocompetent cells and blood vessels.

Noye’s criteria for endometrial dating was considered the gold standard approach for evaluating endometrial responsiveness and detecting endometrial abnormalities [19]. Its disadvantages include

·               Disruption of normal anatomical layering by endometrial biopsy

·               High intra- and inter-observer variability

·               Error in endometrial dating for biopsies taken during the 2 days following ovulation as the morphological features of the endometrium do not change significantly during this period

Ultrasonographic evaluation of endometrial thickness and its echogenic pattern is a non-invasive technique to assess the endometrium. Assessment of endometrial blood flow adds a physiological dimension to the anatomical ultrasound parameters. However, the use of endometrial and sub-endometrial blood flow in the prediction of implantation and pregnancy remains unclear.

Pinopods are bleb-like protrusions found on the apical surface of the endometrial epithelium [20]. These structures are several micrometers wide and project into the uterine lumen above the microvilli level. Pinopod expression is limited to a brief period of 48 h in the menstrual cycle corresponding to the putative window of implantation [2122]. Others have detected that pinopods may be present throughout the mid to late secretory phase, however, displaying cycle-dependent morphological changes. This suggests that functionality, rather than pinopod presence or absence, is of greater significance.

The appearance of pinopods is progesterone dependent, and association between mid-luteal increase of progesterone level and the first appearance of pinopods in the menstrual cycle was noted [23]. The detection of pinopods, by electron microscopy, during the mid-secretory phase may be a useful test for assessment of endometrial receptivity to optimize implantation rates; however, it is an invasive test.

There are several proposed molecular markers of endometrial receptivity (Table 23.1). Integrins are a family of trans-membrane glycoproteins containing extracellular, trans-membranal and intracellular domains. Integrins whose expression is increased in the mid-luteal phase were proposed as markers for the window of implantation [24]. Three cycle-specific integrins are co-expressed by the human endometrium defined histologically on days 20–24 of the human menstrual cycle: α1ß1, α4ß1 and αVß3, but only the ß3 mRNA subunit expression was shown to increase after day 19 and is not detected beforehand [25]. With respect to its expression pattern along with its epithelial localization, aVß3 has been proposed as a potential receptor for embryonic attachment [26].

Table 23.1

Molecular Markers of Implantation

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 [2729], 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 [3436].

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 [4041]. 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 [4647].

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 [5052].

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 [6869]. 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].

The beneficial effects of atosiban were observed not only in patients who had a high frequency of uterine contractions but also in those who had a low frequency. These findings suggest that atosiban may have other benefits and is effective in priming the uterus for implantation, in addition to its effect on contractions of the uterus [77].

Lower dosage of atosiban (a single bolus dose of 6.75 mg) before Et also improves pregnancy outcomes of patients with RIF. A significantly higher implantation rate was found in those receiving atosiban before ET than in those receiving it immediately after ET [78]. More studies are required to find out exactly how atosiban works and to evaluate its role in patients with RIF undergoing IVF.

23.5.8 Neuromuscular Electrical Stimulation (NMES) Therapy

Thin endometrium at the time of ovulation has been demonstrated to be an important factor in implantation failure. Uterine receptivity in women with thin endometrium may be poor due to the impairment of blood flow impedance through the endometrium. NMES is the application of electrical stimulation to a group of muscles through electrodes placed on the skin. NMES was performed 3–4 times for 20–30 min or once a day consecutively in the follicular phase. Pelvic floor NMES was found to significantly enhance endometrial thickness in patients with thin endometrium. Contraindications to NMES therapy include vaginal wall prolapse, skin breakdown around the peri-anal region, rectal bleeding, complete denervation of the pelvic floor, presence of cardiac pacemaker, cardiac arrhythmia, unstable seizure disorder, pelvic pain and painful haemorrhoids [79].

It is possible that NMES corrects the impairment of uterine blood flow impedance. Though the exact mechanism by which NMES exerts its effect on the process of angiogenesis and vascularization in the endometrium is unknown, the increased blood supply towards the endometrial and the sub-endometrial regions may be due to the repeated contraction and relaxation of the uterine smooth muscle. NMES being a safe and non-invasive technique is a promising alternative for managing patients with thin endometrium.

23.5.9 Local Injury to the Endometrium

Local inflammatory reactions are necessary for angiogenesis and a successful implantation. This observation led to the hypothesis that endometrial injury might improve implantation in patients with repeated implantation failure as a result of subsequent inflammatory responses and changes in cytokine production in the endometrium.

Studies have shown that prior incidence of hysteroscopic endometrial biopsy is associated with increased rates of implantation, clinical pregnancy and live birth among women who experienced repeated implantation failure but without obvious endometrial defects. This suggests that a hysteroscopic procedure in the preceding cycle could be beneficial for improving pregnancy in subsequent IVF cycles. There were substantial variations in patient selection, timing, number and extent of endometrial injury applied and techniques in these studies [8084].

In a recent pilot study, it was demonstrated that a site-specific hysteroscopic endometrial injury (a 2 × 2 mm injury at the midline posterior wall about 10–15 mm from the fundus) performed during the ongoing IVF cycle between D2-D7 instead of injuries received during prior cycles significantly improves subsequent embryo implantation in patients with RIF. The endometrium in cycles undergoing endometrial biopsy was found to be thicker; however, the difference did not reach statistical significance [85].

Some of the possible mechanisms by which endometrial injury improves endometrial receptivity include decidualization of endometrium [86] and a wound healing process involving secretion of different cytokines and growth factors beneficial for embryo implantation [87]. Also, synchronization of endometrial and embryo development may play a role as it has been reported that COH cycles result in different structural and functional changes in comparison to natural cycles, including histological advancement, pinopod maturation advancement and steroid receptor down-regulation [88].

However, conflicting results were shown in a Cochrane review, and local injury to the endometrium on the day of oocyte retrieval disrupts the receptive endometrium and has a negative impact on implantation in IVF cycles [89].

23.5.10 GnRH Agonist Injection in Mid Luteal Phase

Administration of a single dose of short-acting GnRH agonist injection 5 or 6 days post ICSI improved the implantation as well as clinical pregnancy rate in all cycles including GnRH antagonist cycles in a meta-analysis of 10 randomized controlled trials, of which 5 trials had usable data for analysis [90]. It has been proposed that GnRH agonist may rescue the corpus luteum by enhancing secretion of pituitary gonadotropins like LH and FSH. However, further studies are required to elucidate the exact mechanism of action.

23.6 Micro RNA Studies

Endometrial receptivity is a complex process involving genetic, morphological and biochemical changes with the expression of numerous molecular mediators. The endometrial gene expression profile changes under the coordinate and sequential action of sex steroid hormones.

Micro RNAs (miRNAs) have emerged as potential regulators of endometrial receptivity and control gene expression at the post-transcriptional level by targeting mRNAs for degradation or translational repression or both. Cell cycle progression, proliferation and differentiation are among the biological processes regulated by miRNAs, processes that are known to occur during the cyclic changes in the endometrium. Luteal support with progesterone and oestrogen + progesterone has a profound effect on endometrial miRNA profiles [91]. Thirteen miRNAs that regulate the expression of 3,800 genes were found to be differentially expressed in secretory endometrium of RIF-IVF patients. Hence, the RIF-associated miRNAs could be exploited as new candidates for diagnosis and treatment of embryo implantation failures [92].

23.7 Embryo Quality

Blastocyst transfer offers higher pregnancy rates and should be offered to all patients with RIF. It is also a well-known fact that in the presence of a receptive endometrium, RIF will occur if the embryos are of poor quality or have aneuploidy [93]. In such cases, pre-implantation genetic diagnosis (PGD) should be offered, and only healthy embryos should be transferred. In the presence of poor endometrial receptivity, the numbers of embryos transferred should be more than single, as the growth factors secreted by one embryo may aid in the implantation of another embryo. In the event of embryos being aneuploid in multiple cycles, the option of donor oocytes may be explored. In the sub-group with normal embryos and non-correctable endometrial compromise, the option of surrogacy may be considered.

Conclusion

The window of implantation is short lived and may be altered in ART cycles due to COH. It may not always coincide with the replacement of a fertilized embryo in the uterine cavity. The process of implantation is complex and as yet poorly understood. It involves two independent variables, the endometrium and the embryo. Both actively secrete integrins, cytokines and growth factors, which are regulated both temporally and spatially in the uterine cavity. The understanding of the regulating mechanisms is still very primitive. Hence, there is a paucity of treatment options available. Most treatment modalities we currently employ are not evidence based, as there is lack of robust randomized controlled trials. The field of genomics, proteomics and metabolomics provides access to a wide variety of genes, miRNA and protein molecules for scrutiny in patients with normal fertility and RIF. However, it has currently not provided the necessary breakthrough in understanding the process of implantation. Research in this field is the need of the day in order to improve the success rates in ART and unexplained recurrent miscarriages.

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