Abstract
With initial attempts of failed IVF, and also after miscarriages, it is quite common for women to assume and become convinced that there is a problem in the uterus or their body leading to the “rejection” of the embryos. This may be true for some women, but it is difficult to detect and predict which women this applies to. In fact, chromosomal errors in the embryos are probably the main underlying reason behind all reproductive failures, but uterine factors may have a role [1]. A recent analysis of more than 15,000 high grade blastocysts showed 30–90% were aneuploid, increasing significantly with a woman’s age [2]. Sometimes pre-implantation genetic screening (PGS) is useful either prior to or in conjunction with adjuvant treatment to assess the necessity and limit the number of embryo transfer cycles and repeated use of adjuvant treatments.
1 Adjuvant Treatment for the Uterus
With initial attempts of failed IVF, and also after miscarriages, it is quite common for women to assume and become convinced that there is a problem in the uterus or their body leading to the “rejection” of the embryos. This may be true for some women, but it is difficult to detect and predict which women this applies to. In fact, chromosomal errors in the embryos are probably the main underlying reason behind all reproductive failures, but uterine factors may have a role [1]. A recent analysis of more than 15,000 high grade blastocysts showed 30–90% were aneuploid, increasing significantly with a woman’s age [2]. Sometimes pre-implantation genetic screening (PGS) is useful either prior to or in conjunction with adjuvant treatment to assess the necessity and limit the number of embryo transfer cycles and repeated use of adjuvant treatments.
While the respective contributions of embryo and endometrium towards reproductive success should be individualised as much as possible, an estimate may be that about 20% of implantation problems are due to the uterus. Women should also be strongly counselled about the known embryo genetic contribution, and it is often helpful to turn around their perception, to illustrate that it is possible that they are highly receptive and not enough selective to embryo implantation, and it is simply that they are unable to detect which embryos to ‘reject’ [1].
2 Endometrial Receptivity Testing: Protein Expression
Endometrial receptor testing is still in experimental stage; this is done at a molecular level analysing the expression of a group of genes related to endometrial receptivity. The testing is done by an endometrial biopsy in the luteal phase of the menstrual cycle and the endometrium is classified as receptive or non-receptive by a computational predictor, which suggests the window of implantation. Rescue of non-receptive endometrium by tailored embryo transfers in a displaced window of implantation resulted in higher pregnancy rate (51.7%) and implantation rate (33.9%) compared to the controls [3].
3 Endometrial Receptivity Testing: Immune Testing
Immune testing has a long and chequered history. With the exception of antiphospholipid antibodies and thyroid antibodies, few tests are widely available in routine clinical practice. The most well-known immune test is for natural killer (NK) cells. The purpose of NK testing is to attempt to be more discerning with immune therapy, so as to only target those who are possibly more likely to benefit from it.
Uterine NK (uNK) cells are rather specialised immune cells in the uterine mucosa participating in implantation, and are likely to play a significant role in ensuring its success [4]. Testing for uNK cells has, so far, primarily been done by immunohistochemistry, which is unable to detect subtypes or function. The enormous increase in numbers during the menstrual cycle (from 5% to more than 40% of all stromal cells) also makes reliable testing extremely difficult [5]. These technical issues explain why studies have been contradictory and, outside of research centres, this kind of testing should, in our opinion, be used with caution.
Peripheral blood NK (pbNK) cells testing has sometimes been dismissed as irrelevant in view of the functional differences of the majority of pbNK cells compared with uNK cells [4]. However, not only is there some evidence of correlation between pbNK and uNK numbers [5] but also pbNK testing can be an independent marker of systemic immune dysfunction. As with uNK testing, technical issues are critical and often underappreciated. However, there is evidence showing higher levels of activated pbNK cells in women with recurrent miscarriage and repeated IVF failure, and hence activated cells may be predictive of IVF success [5].
4 Immune Therapy
There is no evidence that immune therapy is beneficial when applied to all IVF cycles or all women with recurrent miscarriage. But, there is increasing evidence of benefit when given to women selected on basis of both their history and known immune dysfunction [6].
4.1 Corticosteroids
Reproductive failure and lower IVF success rates were noted in women with immune dysfunction. Immune-suppressive activity of corticosteroids was, therefore, expected to improve pregnancy outcome by reducing endometrial pro-inflammatory cytokines production and uNK cell activity [7]. The authors found that corticosteroids did not improve the clinical pregnancy rate in the study population, but subgroup analysis revealed a significantly higher pregnancy rate in the IVF, rather than ICSI group.
In a systematic review [6], there was a higher clinical pregnancy rate noted in the prednisolone group. However, data heterogeneity was substantial, thus suggesting a cautious interpretation of the results.
From various studies, significantly higher pregnancy and implantation rates were noted in women undergoing IVF, with high anticardiolipin antibody (ACA) when treated with methylprednisolone and low-dose aspirin. In presence of antithyroid antibodies (ATA) higher implantation and clinical pregnancy rates were seen in women treated with corticosteroids, aspirin and levothyroxine. In women with high uNK cells and unexplained recurrent miscarriage, live birth rates were higher in the prednisolone-treated group [8].
A number of investigators looking in to the use of steroids alone or in conjunction with other adjuvant treatments in women with positive antinuclear antibody (ANA) undergoing IVF have reported contradictory outcomes. The Cochrane review by Boomsma and colleagues (2012) [9] showed no evidence of improved clinical outcome with pre-implantation administration of glucocorticoids.
This clearly explains that steroids, in isolation or combination, are beneficial when used in targeted groups with immune dysfunction, high ACA, ATA or high uNK cells. Steroids are obviously cheap, easy to administer, widely available and have a fairly extensive history of use. The only known potential caution is the weak association with cleft palate; a threefold increase in oral clefts among offspring of women who received oral corticosteroids during pregnancy, which should be explained to the couple [10].
4.2 Intravenous Immunoglobulin (IVIg)
IVIg is a pooled blood product from numerous donors. Complications or side effects are seen in up to 35% of cases and are often related to the rate of infusion, total dose and brand of IVIg infused. Mild and transient side effects include flushing, headache, itching, low backache, nausea and fatigue. Serious and rare side effects include aseptic meningitis, severe anaphylactic reaction, acute renal failure and thrombotic events. We have previously argued that women should be provided with clear information of these side effects before embarking on the treatment [8].
Polanski et al. (2014) [6] reviewed 217 studies with high pbNK cells and identified only 3 eligible studies for analysis. The findings suggested that immune therapy for women with high pbNK cells is beneficial. Other authors found that, in women with high uNK cells and unexplained recurrent miscarriage, live birth rates were higher when IVIg was used [11]. A systematic review [12] showed significantly lower miscarriage rate, higher implantation rate, clinical pregnancy rate and live birth rate after IVIg treatment. However, the review noted numerous methodological flaws in the studies included, such as poor design, lack of randomisation, heterogeneous population, use of multiple adjuvant interventions, different doses and regimes of IVIg and lack of cost effective analysis.
4.3 Intralipid Infusion
Intralipid infusion therapy has been used for decades to correct fatty acid and calorie deficiency among those who are unable to feed orally. The intralipid infusion is an emulsion of soya bean oil, egg yolk, phospholipids, glycerin and water.
Intralipids function as immune-modulators by inhibiting pro-inflammatory factors such as TH1 cytokines [13]. It was shown that intralipid infusion could effectively suppress NK cells activity in patients with abnormal NK cytolytic activity. It is postulated that the fatty acids within the emulsion serve as ligands to activate peroxisome proliferator-activated receptors expressed by the NK cells, which would reduce NK cytotoxic activity resulting in enhanced implantation and maintenance of pregnancy [14].
A non-randomised trial showed a 46% clinical pregnancy rate with the use of 20% intralipid fat emulsion in women with recurrent implantation failure and elevated TH1 cytokine response on NK cell assay. It was noted that TH1:TH2 activity ratio decreased following treatment and this alteration in cytokine activity was deemed to be responsible for the outcome [15].
The first randomised controlled trial (RCT) with intralipid was recently presented at the ASRM Annual Meeting in Baltimore [16]. Women with repeated IVF failure and abnormal immune testing were randomised to receive intralipid or nothing. With over 100 in each group, the intralipid infusion group had significantly higher live birth rates (32%) than those who did not (12%). Given the relatively low cost and maternal-fetal safety, this type of immune therapy is likely to become more prevalent. However, caution is still necessary, not least because of the potential infection at the cannula site which has led recently to reported death [17].
4.4 Anti-tumour Necrosis Factor-Alpha
TNF-α antagonists suppress inflammatory response to tumor necrosis factor (TNF) and have been used in various autoimmune and immune-mediated conditions such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease and psoriasis. Prolonged use of TNF inhibitors is associated with serious side effects such as lymphoma, demyelinating disease, congestive heart failure, induction of autoantibodies and lupus-like syndrome [18]. Direct correlation between circulating TNF-α levels and IVF outcome has yet not been fully established [8], but its use has so far been directed at women with abnormal T cell activation.
An exaggerated TH1 response is harmful to the process of embryo implantation resulting in infertility. Anti-TNF-α agents, such as Entanercept (Enbrel), Adalimumab (Humira) and Infliximab (Remicaid) were studied in infertile patients with raised TH1:TH2 cytokines ratio undergoing IVF, and a higher live birth rate was noted [19]. This treatment is however expensive and not used widely outside specialised centres.
5 Hormone Therapy: Metformin
Metformin is an insulin-sensitising agent. In women with polycystic ovary syndrome (PCOS), metformin appears to act as a brake on the response of polycystic ovaries to exogenous gonadotrophin stimulation minimising the risk of ovarian hyperstimulation syndrome (OHSS). Side effects, mainly gastrointestinal symptoms, occur in approximately 10% of patients on metformin. There is no evidence of teratogenicity.
An RCT comparing metformin with placebo in women with PCOS undergoing IVF treatment [20] demonstrated a significant increase in the clinical pregnancy rate beyond 12 weeks and a clinically significant reduction in the occurrence of severe OHSS. The Cochrane review [21] showed similar findings in terms of prevention of OHSS. A recent meta-analysis of metformin administration in women with PCOS undergoing ovulation induction with gonadotrophins found increased pregnancy and live birth rates in the treatment group [22]. Interestingly, Brewer and colleagues (2010) [23] concluded that women who took metformin in the fresh cycle of IVF treatment had a significantly higher live birth rate in the subsequent frozen embryo transfer cycle.
6 Improving Uterine Blood Flow
6.1 Aspirin
Aspirin (Acetylsalicyclic acid) is a non-steroidal anti-inflammatory agent (NSAID). Daily administration of low-dose aspirin results in vasodilation and increase in the peripheral blood flow by inducing a shift from thromboxane A2 to prostacyclin [24]. Aspirin was also shown to increase uterine blood flow [25], which in turn enhances the endometrial receptivity resulting in higher embryo implantation rates. However, these findings were not shown to translate into clinical use.
Aspirin was shown to be beneficial in women with recurrent miscarriage and antiphospholipid antibody syndrome (APS) as well as in prevention of early onset pre-eclampsia (CLASP Study). The Cochrane review [26] confirmed that administration of aspirin in IVF does not improve pregnancy rates. There was no significant difference noted between aspirin and control group for the live birth rate, clinical pregnancy rate or miscarriage rate.
6.2 Heparin
Heparin is an anticoagulant. It improves implantation by regulating the endometrial receptivity and decidualisation of endometrial stromal cells by several means.
In women with thrombophilia, impaired trophoblastic invasion of maternal vessels by the syncytiotrophoblast is noted due to micro-thrombi at the site of implantation [27]. In this group of women, heparin could improve implantation, however, published studies showed contradictory findings. Significant differences in pregnancy rates were noted in women with thrombophilia receiving heparin treatment with or without low-dose aspirin and no improvement in pregnancy rates when unfractionated heparin and low-dose aspirin were used [8]. A Cochrane systematic review [28] showed a 77% increase in live birth rate, 73% in implantation rate and a 78% reduction in miscarriage rate. The same review also found a 79% increase in live birth rate in women with history of three recurrent implantation failures (RIF). Interestingly, a similar improvement was not seen when studies included only unexplained RIF.
6.3 Vasodilators
Endometrial development plays a pivotal role in successful embryo implantation. Increased endometrial blood flow and thickness are likely to improve implantation and IVF success rates. Several agents have been tried to improve sub-endometrial blood flow and increase endometrial performance during IVF treatment.
6.3.1 Vasodilators: Nitric oxide (NO) and Nitroglycerine (NTG)
Nitric oxide is a vasodilator and regulates the smooth muscle of blood vessels. NO plays a significant role in decidualisation and implantation. NO production inhibitor in the post-ovulatory phase has been associated with pregnancy failure. Nitroglycerine (NTG) is an NO donor, and has been investigated to assess the efficacy in inducing uterine vasodilation and thereby endometrial receptivity. It was recently summarised that there was no significant difference in the implantation and pregnancy rates or the uterine artery Doppler between the placebo and the NTG groups [8].
Vasodilators: Sildenafil Citrate
Sildenafil citrate (Viagra) potentiates the effect of NO on vascular smooth muscle. Sildenafil improved radial artery resistance index, reduced uterine artery pulsatility index (PI) and endometrial thickness, and enhanced pregnancy rates in some studies, but similar findings in women undergoing either fresh IVF cycles or frozen embryo transfers were not noted [8].
7 Uterine Relaxants
The uterine smooth muscle contractility varies throughout the course of the normal menstrual cycle. The uterine activity in the IVF cycles is higher compared with the natural cycle conception, a phenomenon which is attributed to various factors including mechanical stimulation with the speculum, embryo transfer catheter stimulating the uterine wall, transfer in the early luteal phase and supraphysiological hormonal milieu. Increased contractility can result in an ectopic pregnancy. Various uterine smooth muscle relaxants are studied in an attempt to optimise IVF success rates and improve success [8].
7.1 Nitroglycerine (NTG)
NTG is an NO donor. It acts as a vasodilator and relaxes the smooth muscle of the uterus. However, use of NTG three minutes before embryo transfer did not show any significant difference in the ease of transfer or the pregnancy rates [29].
7.2 β2-Adrenergic Antagonists
Selective β2-adrenergic blockers (Ritodrine, Terbutaline and Salbutamol) are known uterine smooth muscle relaxants used in obstetrics for management of pre-term labour and before external cephalic version. Administration of these agents regularly for two weeks in the luteal phase, following oocyte retrieval did not show any improvement in the implantation and pregnancy rates [30], whilst resulting in adverse side effects such as hypotension and tachycardia.
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