Assessment of CD56+ CD69+ Natural Killer Cells in the Peripheral Blood of Women with Repeated Implantation Failure (RIF) undergoing an Intra Cytoplasmic Sperm Injection (ICSI) Cycle

Abstract

Recurrent implantation failure (RIF) is the failure of implantation of the embryo into the uterine wall following embryo transfer in an ICSI cycle. The ‘recurrent’ element is usually defined as three failed ICSI cycles, however this varies among literature and studies undertaken have often limited this definition to at least two failed ICSI. High number of immune cells, such as NK cells, regulatory T cells and macrophages, accumulate in decidua during the early phase of gestation and are required for a successful pregnancy. NK cells are found in both peripheral blood and the uterine mucosa. There are, however, important phenotypic and functional differences between NK cells present at the two sites. CD molecules are commonly utilized in cell sorting techniques and cell populations are usually defined as either positive or negative; they control inhibition and activation; proliferation and effector functions (cytotoxicity and cytokine production). CD56dim cells are potent mediators of natural cytotoxicity and more cytotoxic than other NK subsets. These cells have direct contact with chorionic villi at the intervillous space. CD69 is one of the earliest cell surface activation markers expressed by NK cells and supportive evidence is provided by in vitro models demonstrated that activated CD69 positive NK cells are capable of lysing trophoblasts. Therefore, this study was conducted to assess the level of CD56+dim and CD69+ in women with repeated implantation failure undergoing an ICSI cycle to be used as as a predictor for successful implantation in ongoing ICSI cycles. This study is a case control study which was conducted at Ain Shams Maternity Hospital IVF center through the period from January 2014 to January 2015. 60 women were included and divided into two groups: The study group included 30 women with history of unexplained infertility and repeated implantation failure in their ICSI attempts and a control group which included 30 normal, non-pregnant and fertile women. A written informed consent was taken from each patient before starting the research and after explanation of the nature, scope and possible consequences of the study. The patients’ data including personal, obstetric, menstrual, contraceptive, medical, surgical and infertility history, clinical examination and investigations were recorded in a specially prepared sheet. Blood samples were obtained during mid luteal phase of the menstrual cycle, which was known by self-reporting regarding to control group and by operator reporting regarding to cases group. Blood samples incubated for 15 min with monoclonal antibodies: anti-CD56 phycoerythrin (PE) and anti-CD69 –fluorescein isothio- cyanate (FITC), Cells were lysed and washed prior to analysis and Flow cytometric analysis of cells was performed. The current study revealed that there was a highly statisticaly significant difference between control and case groups regarding CD3- CD56+dim NK percentage with a p-value <0.0001. The current study revealed that there was a statistically significant difference between control and case groups regarding CD3- CD56+dim CD69+ NK cells percentages with a p-value 0.003. Our study also revealed that CD56+dim NK had a very good predictive value as evidenced by an area under the ROC curve (AUC) of 0.79 (95% CI, 0.68 to 0.90; p-value, <0.0001). This had a sensitivity of 100%, a specificity of 43.3%, a PPV of 63.8%, and a NPV of 100%. This study also report that, CD56+dim CD69+ NK cells had a very good predictive value as evidenced by an AUC of 0.74 (95% CI, 0.61 to 0.87; p-value 0.0003). This had a sensitivity of 86.7%, a specificity of 63.3%, a PPV of 70.3%, and a NPV of 82.6%.