All we need to know about N K cells which are detrimental for balstocyst

A to Z of N KCells:

A to Z of N KCells:-Women with larger populations of cytotoxic CD16+ eNK cells(endometrial N K Cells)  may be at greater risk for infertility disorders and endometriosis . This is an effect of inflammatory influences of endometriosis. CD16+ NK cells display cytotoxic activity towards trophoblasts and endometrial cells.S

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 Routine  assessment of NK cells not recommended. But endometrial natural killer cells (eNK) are the most abundant leukocyte population in the endometrium and have been thought to play a key role in the etiopathogenesis of endometriosis and reproductive disorders. CD56 bright  CD16- NK cells can produce angiogenic factors that promote spiral artery remodeling and cytokines (LIF, leukaemia inhibitory factor) that direct the migration of trophoblasts. Their function is controlled by inhibitory receptors (NKG2a) and activating receptors (NKp46).

: Women with larger populations of cytotoxic CD16+ eNK cells may be at greater risk for infertility disorders and endometriosis.

Further, the difference between infertility and recurrent loss might represent a continuum of changes related to the inflammatory influences of endometriosis. This is a study I am quoting.

One more interesting article....Pregnancy has been reported to be associated with shift away from T-helper-1 (Th1)–type and bias toward T-helper-2 (Th2)–type immune responses . The Th1/Th2 concept has been extended by demonstrating that natural killer (NK) cells also can show comparable polarities in their cytokine secretion profiles . The cytokine repertoire of peripheral blood NK cells is mainly type 1 cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α. However, NK cells can be induced with stimulation to produce type 2 cytokines, such as interleukin (IL)-4, IL-5, and IL-13 . There are additional types of NK cells, which produce transforming growth factor-β (5) or IL-10 (NKr1) . It has been proposed that cytokine production by NK cells facilitates decidualization, controls trophoblast invasion, and promotes angiogenesis at the implantation site .

In peripheral blood, NK cells are either CD56bright or CD56dim cells, the main population being CD56dim NK cells. CD56bright cells are mainly present in the decidua although a relatively small population is present in the circulation. Recently, it has been reported that the type 2 shift during pregnancy is predominantly in the NK-cell (CD56bright and CD56dim) and NKT-cell (CD56+CD3+) populations instead of in the T-helper or cytotoxic T-cell populations .Type 2 shift is reported to be related to a significant decrease in type 1 cytokine production with no change in type 2 cytokine production by NK cells after in vitro stimulation in healthy pregnant women (10). Contrary to type 2 shift in normal pregnancy, the type 1 shift was present in women with pre-eclampsia predominantly in the NK cell populations.

Studies suggest that systemic regulation of peripheral blood NK cells is indicative of reproductive success, and implantation-related immune abnormalities, such as uterine NK cells, cytokines, and leukemia inhibitory factor, contribute to reproductive failure. The increase of cytotoxic NK cells in peripheral blood and endometrium was related negatively to the therapeutic results of IVF-ET . In women with recurrent SAB or repeated IVF-ET failure, peripheral blood NK cells have higher proportions of activated NK cells in vivo with unbalanced CD69 and CD94 expression .

A propensity to Th1 immune responses has been reported in women with recurrent SAB and implantation failures after IVF-ET systemically or locally. The presence of elevated Th1/Th2 cell ratios, high concentration of Th1 cytokines secreted by peripheral blood mononuclear cells (PBMCs), elevated NK cell cytotoxicity and levels, and emergence of various autoantibodies are the supporting evidence .Basically natural killer cells are important markers and have two sets of cells one is implantation friendly and another not so, but measuring their level in blood is not as beneficial as it would have been in endometrial tissue as these cells are very few in number and difficult for flow cytometry to catch it . Until unless some better tests are available we can't do it routinely.

Self collected samples is portable and does not require running water or electricity gives rapid results in 2.5 hours thus allowing screening and follow up treatment in a single visit.

Self collected samples is portable and does not require running water or electricity gives rapid results in 2.5 hours thus allowing screening and follow up treatment in a single visit. This  new test has been developed for screening women in developing regions for cervical intraepithelial neoplasia by detecting 14 high risk hpv types. This test is simples and can be performed even by health care workers. It works on self collected samples is portable does not require running water or electricity gives rapid results in 2.5 hours thus allowing screening and follow up treatment in a single visit. It will soon be commercially available. The evaluation of clinical accuracy of care hpv as a rapid screening test in rural china for the detection of high grade cervical neoplasia showed it to be substantially better than via with sensitivities and specificities of 90.0% and 84.2% respectively on cervical specimens collected by nurse midwife and 81.4 % respectively on self collected vaginal specimens.

Efforts are underway to improve screening coverage in populations where accessibility of screening procedures or its acceptance due to social cultural factors is limited. Several studies have evaluated the diagnostic accuracy of self collected vaginal specimens using swabs brushes etc for hpv. The studies how good acceptance among women. Hence this could be valuable screening method for women who refuse to attend clinic based screening . According to a meta analysis the sensitivity of self collected vaginal specimens for hpv was 74 5 ans specificity was 88% . Clinician obtained specimens had slightly better test characteristics as compared to self collected specimens . AUK trial found similar sensitivity between clinician and self hpv tests for women with high and low grade disease. However self hpv test missed few high grade disease as compared to the clinician sample though the difference was not significant. A RCT comparing hpv screening by self sampling with clinic based cervical cytology in Mexican women found relative sensitivity of hpv testing to be 3.4 times greater with 4.2 times more detection of invasive cancers than cytology.

The persistence of oncogenic hpv is considered to be true precursor of cervical cancer. The transcription of the viral oncogenes E6 and E7 is necessary for the malignant transformation and maintenance of th neoplastic state. Several studies indicate that the up regulated expression of hpg E6/E7 genes is necessary for the initiation and progression of cervical neoplasia. A new test indentifying E6/E7 mrna as biomarker is under development which will help in differentiating women who have hpv infection from those who have begun to develop neoplastic precancerous cells studies show this test to be highly sensitive yet significantly more specific for cervical disease compared to hpv dna assays . This test has the potential to be reliable for both primary cervical cancer screening and the triage of borderline cytological abnormalities.

How useful will be Human Papilloma virus testing in the prediction of HGSIL, AGS?? :

So will `HPV based screening test help tye community as a whole??

The identification of strong causal  relationship between persistent infection of the genital tract with high risk hpv types and occurrence of cervical pre cancer and cancer has resulted in the development of a number of screening tests based on hpt dna or rna detection systems. The four possible clinical applications of detecting high risk hpv dna are 1) as a primary screening test solely or in combination with cytology to detect cervical cancer precursors 2) as triage for women with cytology findings of ascus or lsil in order to select women who need referral for colposcopic diagnosis and treatment 3) in subsequent management of women referred for colposcopy due to abnormal smears but where findings on colposcopy /biopsy are negative and 4) as a follow up test for women treated for high grade cin with local ablative or excisional therapy in order to identify rapidly and accurately evaluate the treatment outcomes.

Hpv tests rely on molecular technologies that detect hpv dna in cervical /vaginal samples collected either by health care provider of self sampling. Hybrid capture technology is the most commonly used. Results from various studies highlight the accuracy of hybrid capture 2 for detecting high grade lesions . Results from one meta analysis with HC2 being used for primary screening demonstrated 23 % more detection of  cin 2 cin 3 or cancer compared to cytology at cut off ascus or lsil  but was 6 % less specific combined hpv and cytology screening results identification of further 4 % more cin 3 lesions but at the expense of 7 % loss in specificity, The pooled sensitivity of hc2 for finding underlying hsil was 89.3% but varied over as large range while the pooled specificity of hc2 in excluding hsil was 87.8% . Both the pooled sensitivity and specificity were higher for trials from north America and Europe as compared to trials from developing countries,.

HPV dna testing is considerably more sensitive but somewhat less specific than cytology at detecting high grade cin . The lower specificity is primarily due to the detection of transient infections that have not produced cytologic changes . Consequently it is suggested that hpv dna test which is more sensitive should be applied first to identify the hpv positive women . This should be followed by cytology which is a more specific test to determine their management. Managing hpv positive but cytology negative women is challenging current evidence suggest repeating screening with both cytology and hpv after one year for such women.

Several studies have shown that HPV negativity alone or in combination with negative cytology signifies a longer disease free interval against cin2+ than being negative for cytology alone. An over view of several meta analysis ans systematic trial reviews shows minimal over diagnosis from HPV testing for women aged over 30 years and the screening internal can be safely extended to at least 6 years with HPV DNA  testing for HPV negative  women.

The follow up results of a cluster randomised trial involving single round of screening in low resource setting demonstrates significant reduction in the numbers of advanced cervical cancers and deaths from cervical cancer with hpv testing. The investigators found it to be most objective and reproducible of all cervical screening tests and less demanding in terms of training and quality assurance. They concluded that with the availability of simple affordable and accurate hpv test it can be used as a primary screening approach in low resource settings for women who are at least 30 years of age.

Molecular techniques to detect hpv dna involve technologies that do not use amplification such as nucleic acid probe tests and those that utilize amplification such as polymerase chain reaction . Further amplification techniques are of three types target amplification target nucleic acids are amplified signal amplification signal generated from each probe is increased by a compound probe or branched probe technology and probe amplification probe molecule itself is amplified . Target amplified hpv assays amongst which pcr is the most common have capacity to detect very small amounts of hpv dna . In this method highly concentrated samples of a specific dna genetic sequence are produced which are then probed to identify the specific hpv genotypes present. PCR is usually inappropriate for large screening programmes in low resource setting because of the considerable skills equipment and costs involved in the procedure.

HPV virus and the leading cause of Ca Cx!!. But can we identify and combat the co factors?? How many of us believe that genital HPV infection is the most common viral sexually transmitted infection and affects roughly 80 % of sexually active people.

What are  oncogenic HPV?? :- Most of us are aware that HPV has an immense role in the etiology of cervical neoplasia and later Cancer cervix -15 genotypes of carcinogenic hpv causes almost all cases of cervical cancer.Chronic infection with oncogenic HPV is a necessary but insufficient cause for the development of cervix cancer. Cervical cancer arises through a series of four steps HPV transmission  1) viral persistenceà 2)  progression of a clone of persistently infected cellsà  to 3)  pre cancer and finally à 4)  then and then invasion. Presence of co factors is very important A)  as high parity B) smoking C)  nutritional deficiency D)  hormonal contraceptive use and E)  presence of other sexually transmitted infections increase the risk. Genital HPV infection is the most common viral sexually transmitted infection and affects roughly 80 % of sexually active people. In most cases HPV infection is cleared by the cell mediated immune system within 1-2 years of exposure. The median time of clearance of HPV infections detected during screening studies is 6-18 months. The small proportion of carcinogenic infections persisting for several years is strongly linked to a high absolute risk of diagnosis of pre cancer. The average time between HPV infection and pre cancer is about 7-10 years . Both mild and moderate dysplasia are more likely to regress than to progress. There is only 1 % risk per year of progression from mild to severe dysplasia or worse but the risk of progression from moderate to severe dysplasia is 16 % within 2 years and 25 % within 5 years.

 More than 100 hpv types have been characterised molecularly and about 40 are known to infect the genital tract. Persistent infection with one of the 15 genotypes of carcinogenic hpv causes almost all cases of cervical cancer. Seventy percent of cervical cancers and about 50 % of cervical intraepithelial neoplasia be attributed to two most carcinogenic hpv types , hpv 16 and hpv 18 while HPV 6 NS HPV 11 are responsible for about 90 % of genital warts,

Most of the pre malignant and malignant lesions are of the squamous type but around 15 % are of the glandular type hpv 16 is associated to a greater extent with squamous cell carcinomas while hpv 18 to adenocarcinoma, Hence in women with persistent hpv 18 findings even in the absence of other cytologic or colposcopic abnormalities a thorough examination of the endocervix should be conducted to exclude hidden lesions. Hpv is found in more than 90 % of cervical cancers 94.7 % of the cervical cancers and 84.4 % of the HSILs among women in rural western india were positive for hpv infection . Presence of hpv express the oncogenic proteins E6 and E7 that inactivate the host regulatory proteins p53 and pRb respectively . Worldwide interest has grown in the potential for hpv testing in cervical cancer prevention programmes.

Is it true that VIN is more sensitive 80 % sensitivity and a 92 % specificity for VIA than Pap smear based cytology?? How helpful is “Visual based screening methods:??:--

The evidence bases in support of has emerged from several studies conducted in different developing countries demonstrating comparable or greater sensitivity of visual inspection of the cervix with naked eye than that of cytology . In addition medical as well as paramedical staff including primary health care workers can be easily trained in the visual inspection techniques in a relatively short period of time. A) By  3-5 % dilute acetic acid:-- Application of 3-5 % dilute acetic acid on the cervix during VIA results in reversible coagulation of cellular proteins. The areas with dysplasia or invasive cancer undergo maximal coagulation due to large number of undifferentiated cells in the epithelium that have a reversal of nuclear cytoplasmic ratio. Therefore these areas appear acetowhite .B) Lugol’s iodine on the cervix:--   During the VILI  procedure after application of Lugol’s iodine on the cervix the normal squamous epithelium containing glycogen takes p iodine staining mahogany brown or black . The precancerous cells and invasive cancer do not take p iodine due to lack of glycogen and appear as well defined thick mustard or saffron yellow areas .

The test characteristics of VIA have been evaluated from several cross sectional studies in India , Africa and china wherein the sensitivity range from 67% to 79 % and the specificity range from 49 % -86 % . A recently published metaanalysis reports 80 % sensitivity and a 92 % specificity for VIA . Several studies record higher sensitivity of VILI as compared to VIA however not the specificity . The sensitivity and specificity in a pooled analysis of eleven cross sectional studies were 76.8 % and 85.5 % and 85.4 % .

If results of randomised controlled trials show reduction in the disease specific mortality then that’s the strongest evidence of effectiveness of a screening test. The results of cluster randomised controlled trial in southern India after a single round of screening using VIA followed by treatment in the same visit when appropriate show a significant 25 % reduction in cervical cancer incidence and a significant 35 % reduction in cervical cancer mortality at the end of seven years of follow up . Another cluster randomised controlled trial of cervix cancer screening in Mumbai India demonstrated a significant down staging of cervix cancers in the intervention arm . Though a single round of VIA screening did not show decrease in the incidence of advanced cervical cancer and significant mortality reduction after eight years of initiation of the osmanabad trial the same may be evident after few more years of follow up .

The Mumbai cross sectional study concludes that parallel testing with both VIA and VILI should be considered where good quality cytology is not leasable and that the sensitivity of cytology and hpv testing can be significantly increased by adding the visual test. The visual inspection procedures require minimal health care infrastructure and the results are available immediately for initiating treatment at the same visit. This single visit approach is especially important to increase the programme effectiveness in the developing countries scenario with high rates of loss to follow up . Visual tests are not reliable in postmenopausal women because of changes in the transformation zone of the cervix the area in which precursors of cervical cancer arise.

DScreening for Ca Cervix:-Cytology based screening programmes are labour intensive and logistically burdensome

Cytology based screening programmes are labour intensive and logistically burdensome. They require multiple visits by the women for various reasons like screening obtaining the results follow up investigations and treatment in cases of abnormal smears . Many programmatic aspects need to be taken into consideration for the programme to achieve the maximum potential public health benefit such as compliance of the women to the screening procedures obtaining an adequate smear transport of samples to the nearest secondary or tertiary care facility for further processing and analysis. Thus despite the low consumables cost high quality cytology is expensive in absolute terms and may not necessarily be the most cost effective option for screening several new technologies like the liquid based cytology and the automated pap smears are being explored.

In liquid cytology the cells are collected using a very small brush that is broken off into a small pot containing preservative solution. In  the cytology laboratory the sample is filtered or centrifuged to remove excess blood and debris and the cells are then transferred to the slide in a mono layer thus obtaining uniformity of the cell  population in each sample. It has logistical and operational advantages such as interpretation at higher speed lower rate of unsatisfactory smears and possibility of ancillary molecular testing using remnant fluid. LBC is more expensive than conventional cytology and requires additional supplies and sophisticated equipment. In a meta analysis comparing conventional pap with LBC no difference was found in the relative sensitivity. Similarly no difference was found in the relative specificity when high grade squamous intraepithelial lesions and low grade squamous intraepithelial lesions were considered as cutoff .  But a lower pooled specificity was found for LBC when presence of atypical squamous cells of underdetermined significance and above were included .

In Automated pap smears testing the material on the slide is reviewed and scored based on an algoilthm . This includes variety of visual characteristics such as shape and optical density of the cells as to the likelihood of an abnormality being present . Auto pap selects a sample of slides for manual re screening that are enriched with abnormalities thereby including most of the slides that exceed a certain threshold for the likelihood of abnormal cells in contrast to random rescreening In autocyte screen a human reviewer after looking at the various cell images that are presented determines whether a manual review is required. The reviewer needs to enter an opinion . if the findings of both the reviewer and the computer are reported as normal then no further review is needed and the diagnosis is reported as within normal limits . Manual review is undertaken for cases which are designated by either the cytologist or the computer ranking as abnormal.

Visual Inspection Methods

It has been difficult to establish and maintain effective cervical cytology programs in developing countries due to lack of resources trained man power infrastructure and the requirement of multiple visits. Hence alternative low cost and effective screening methods based on visual examination of the cervix that require simple equipment and relatively brief training have  been explored for the control of cervical cancer in low resource settings.

How to assess the effectiveness of a screening test for Cancer of the uterine cervix ?? Where is the evidence??

Organized cytology based screening programmes and the availability of accessible and good quality diagnosis and treatment facilities. Each screening test has its own strengths and limitations. Precancerous lesions are mostly asymptomatic. Limited sensitivity of conventional pap cytology ranging between 30 % and 87% . This is an area of major concern as high false negative rates results in premalignant or malignant cells being misdiagnosed as normal.

India alone accounts for one fourth of the global cervix cancer burden. Cancer of the uterine cervix is the second most common cancer among women globally. An estimated 550,700 new cases and 286, 823 deaths due to cervix cancer are estimated to have occurred in the year 2010 . More than 85 % cases and 885 deaths from cervix cancer occur in developing countries where women often in developing countries where women often lack access to cervical cancer screening and treatment.  .

This large scale morbidity and mortality is in warranted for two important reasons. Firstly the disease develops slowly after initial infection with the human papilloma virus and secondly unlike most other cancers it is preventable when precursor lesions are detected and treated. Women often do not experience any symptoms until the disease has advanced . Hence detection of cervix pre cancers and early stage cancers is  possible only through screening . The morbidity and mortality due  to cervix cancer declined dramatically in high income countries after introduction of organized cytology based screening programmes and the availability of accessible and good quality diagnosis and treatment facilities. In contrast even today millions of women in the developing countries are never screened for cervical cancer in their entire life time ; This is dues to lack of knowledge inaccessibility and poor quality of cervical cancer prevention and control services.

Several tests have been developed to screen women for cervix pre cancers and cancers . Each screening test has its own strengths and limitations. The same modality for cervix cancer control whether it is A) cytology visual based screening B) HPV DNA testing or C) HPV vaccination will never meet the demands of populations throughout the world .In the underserved populations factors such as low cost fewer visits for screening are vital. The choice of the test will depend on its technical performance cost effectiveness the available resources and the socio cultural settings in which it is to be used the various cervix cancer screening tests are described here.

Different Cervix cancer screening methods

A)Cytology—Based  screening.

A)Conventional cytology based screening with Pap smear test developed by George Papanicolaou has been the mainstay of cervical cancer prevention worldwide since the 1950s . Pap test has been exceptional to be accepted in a screening programme without its efficacy being tested with rigorous randomized controlled trials. However now there is convincing evidence about the benefits of cytology screening from many developed countries that introduced Pap test several decades ago. These countries are witnessing reduction in the incidence and death rates from cervix by over 50 % by screening with Pap test.

Cytology based screening programmes can be implemented effectively only if infrastructure and laboratory quality assurance requirements are consistently met.

Various studies have demonstrated limited sensitivity of conventional pap cytology ranging between 30 % and 87% . This is an area of major concern as high false negative rates results in premalignant or malignant cells being misdiagnosed as normal .Consequently the test needs to be repeated at frequent intervals to achieve programmatic effectiveness pap test has repeatedly demonstrated good specificity ranging from 86% to 100% .