Asif, Sonia
(2019)
The significance of natural killer cells in embryo implantation during in vitro fertilisation cycles.
PhD thesis, University of Nottingham.
Abstract
A delay in achieving pregnancy is a global challenge affecting 1 in 7 couples (Evers 2002). The wide accessibility of in vitro fertilisation(IVF) has revolutionized the treatment of infertility and led to the birth of 8 million babies worldwide (van Loendersloot, Repping et al. 2014, De Geyter, Calhaz-Jorge et al. 2018).
Despite this accomplishment, unsuccessful cycles and in particular implantation failure remain a challenging clinical diagnosis for patients and fertility specialists (Simon and Laufer 2012, Coughlan, Ledger et al. 2014). This has led to the introduction of numerous clinical tests aimed at testing the immune, anatomical and genetic components of implantation with very little scientific rationale (Kwak-Kim, Han et al. 2013).
The most widely available immunological investigation is the natural killer (NK) test (Tang, Alfirevic et al. 2011, Kwak-Kim, Han et al. 2013, Seshadri and Sunkara 2014). The interest in NK cells is supported, by the observation that they comprise around 70% of the immune cells in the late secretory phase and first trimester endometrium (Moffett-King 2002, Bulmer and Lash 2015). They may also have a specific function in orchestrating angiogenesis and vascular modelling of the early placenta (Quenby, Nik et al. 2009) along with tolerance of the hemi-allogenic fetal graft (Zhang, Dunk et al. 2016). Furthermore many cohort studies have shown NK cells are raised in women who have unsuccessful IVF treatment (Coulam and Goodman 2000, Tuckerman, Mariee et al. 2010, Sacks, Yang et al. 2012). The mechanism whereby this occurs and its significance is still poorly understood (Lash and Bulmer 2011).
NK testing involves measuring their numbers in either an endometrial biopsy and/or peripheral blood (PB) sample. There are currently no national guidelines on when this test should be offered and the reference range that constitutes an abnormal result (Tang, Alfirevic et al. 2011, Templer and Sacks 2016). Furthermore a range of immune therapies have emerged to treat raised NK results and promote implantation (Polanski, Barbosa et al. 2014). These interventions include oral steroids, intravenous immunoglobulin and intralipid (Alecsandru and Garcia-Velasco 2015, Nardo, El-Toukhy et al. 2015). There is uncertainty as to whether these potentially harmful drugs should be offered to patients when no clear relationship or reference ranges for abnormal NK levels exist (Rai, Sacks et al. 2005, Moffett and Shreeve 2015, Sacks 2015).
There are three main studies presented in this thesis with the aim of investigating the role of NK cells in IVF. One of the main strengths of this work is that women were recruited from a highly successful IVF clinic .The clinical pregnancy rates are around 10% higher than the national average (HFEA 2018). Therefore if IVF did not result in a pregnancy, then endometrial receptivity or adverse maternal immunology could be valid contributory factors.
The first study was a prospective analysis of 203 patients who had endometrial NK testing through immunohistochemistry (IHC) and subsequently embarked on IVF treatment. The main objective was to establish the prevalence of raised NK status in women undergoing IVF. This was coupled with a retrospective analysis of previous IVF and pregnancy history to identify features that were predictive of a raised result. The prevalence of raised NK status was high in this cohort of women (greater than 60%). This could be a true representation but is likely to be flawed due to timing the biopsy without LH testing and sample size. The only predictive demographic factors that showed statistical significance with raised NK status were age and embryo quality.
The second study involved the application of a comprehensive flow cytometry (FC) panel to paired PB and endometrial samples, taken from 42 women undergoing IVF treatment. This technique demonstrated a detailed analysis of NK cells and other immune cell populations; NKT, CD8+ and CD4+ which may have a role in implantation. A functional analysis of these populations with the measurement of inflammatory and angiogenic cytokines; perforin, granzyme, tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) was also conducted. The main finding was that the expression of perforin and granzyme was noticeable in the PB samples as opposed to significant levels of TNFα and IFNγ in the endometrium.
Correlation of the panel with clinical outcomes revealed no differences in the immune cell populations present in the PB and endometrium. However the expression of PB NKT perforin, co- expression of perforin+granzyme+ along with endometrial CD8+ TNFα+IFNγ+ were statistically correlated with live birth. Furthermore a shift in the expression of PB perforin+granzyme+ from a low percentage in those with a live birth to a higher value in patients with a negative pregnancy test was observed.
The third study was a large systematic review of 10 immune-therapies given in the context of unexplained implantation failure to determine if there was an improvement in live birth. This review highlighted that immune therapies were given without any risk assessment of side effects. Although there was some weak evidence to support the use of intralipid and heparin, the finding have to be interpreted with extreme caution.
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