Prolonged SARS-CoV-2 T Cell Responses in a Vaccinated COVID-19-Naive Population.

INTRODUCTION: Exploring T cell response duration is pivotal for understanding immune protection evolution in natural SARS-CoV-2 infections. The objective of the present study was to analyze the T cell immune response over time in individuals who were both vaccinated and COVID-19-naive and had undetectable levels of SARS-CoV-2 IgG antibodies at the time of testing. METHODS: We performed a retrospective descriptive analysis using data extracted from the electronic medical records of consecutive adult individuals who underwent COVID-19 immunity screening at a private healthcare center from September 2021 to September 2022. The study participants were divided into three groups according to the post-vaccination time period, as follows: group A (up to 3 months), group B (3-6 months), and group C (>6 months). T cell response was evaluated using the IGRA methodology T-SPOT®.COVID. RESULTS: Of the total number of subjects (n = 165), 60/165 (36.4%) had been vaccinated in the last 3 months (group A), 57/165 (34.5%) between 3 and 6 months (group B), and 48/165 (29.1%) at least 6 months prior to the examination day (group C). T cell positivity was reported in 33/60 (55.0%) of group A, 45/57 (78.9%) of group B, and 36/48 (75%) of group C (p < 0.007). No statistically significant differences were revealed in the spot-forming cell (SFC) count among groups, with mean SFC counts of 75.96 for group A, 89.92 for group B, and 83.58 for group C (Kruskal-Wallis test, p = 0.278). CONCLUSIONS: Our findings suggest that cellular immunity following SARS-CoV-2 vaccination may endure for at least six months, even in the presence of declining or absent IgG antibody levels.

Assessment of artificial intelligence model and manual morphokinetic annotation system as embryo grading methods for successful live birth prediction: a retrospective monocentric study.

PURPOSE: The introduction of the time-lapse monitoring system (TMS) and the development of predictive algorithms could contribute to the optimal embryos selection for transfer. Therefore, the present study aims at investigating the efficiency of KIDScore and iDAScore systems for blastocyst stage embryos in predicting live birth events. METHODS: The present retrospective study was conducted in a private IVF Unit setting throughout a 10-month period from October 2021 to July 2022, and included the analysis of 429 embryos deriving from 91 IVF/ICSI cycles conducted due to infertility of various etiologies. Embryos incubated at the Embryoscope+ timelapse incubator were analyzed through the established scoring systems: KIDScore and iDAScore®. The main outcome measure was the comparison of the two scoring systems in terms of live birth prediction. Embryos with the higher scores at day 5 (KID5 score/iDA5 score) were transferred or cryopreserved for later use. RESULTS: Embryos with high KID5 and iDA5 scores positively correlated with the probability of successful live birth, with KID5 score yielding a higher efficiency in predicting a successful reproductive outcome compared to a proportionally high iDA5 score. KID5 demonstrated conservative performance in successfully predicting live birth compared to iDA5 score, indicating that an efficient prediction can be either provided by a relatively lower KID5 score or a relatively higher iDA5 score. CONCLUSION: The developed artificial intelligence tools should be implemented in clinical practice in conjunction with the conventional morphological assessment for the conduction of optimized embryo transfer in terms of a successful live birth.

Persistence of T-Cell Immunity Responses against SARS-CoV-2 for over 12 Months Post COVID-19 Infection in Unvaccinated Individuals with No Detectable IgG Antibodies.

BACKGROUND: Immune response to SARS-CoV-2 is crucial for preventing reinfection or reducing disease severity. T-cells' long-term protection, elicited either by COVID-19 vaccines or natural infection, has been extensively studied thus far; however, it is still attracting considerable scientific interest. The aim of the present epidemiological study was to define the levels of T-cellular immunity response in a specific group of unvaccinated individuals from the general population with a prior confirmed COVID-19 infection and no measurable levels of IgG antibodies. METHODS: We performed a retrospective descriptive analysis of data collected from the medical records of consecutive unvaccinated individuals recovered from COVID-19, who had proceeded to a large private medical center in the Attica region from September 2021 to September 2022 in order to be examined on their own initiative for SARS-CoV-2 T-cell immunity response. The analysis of T-cell responses was divided into three time periods post infection: Group A: up to 6 months; Group B: 6-12 months; Group C: >12 months. The SARS-CoV-2 T-cell response was estimated against spike (S) and nucleocapsid (N) structural proteins by performing the T-SPOT. COVID test methodology. SARS-CoV-2 IgG antibody levels were measured by the SARS-CoV-2 IgG II Quant assay (Abbott Diagnostics). RESULTS: A total of 182 subjects were retrospectively included in the study, 85 females (46.7%) and 97 (53.3%) males, ranging from 19 to 91 years old (mean 50.84 ± 17.2 years). Among them, 59 (32.4%) had been infected within the previous 6 months from the examination date (Group A), 69 (37.9%) had been infected within a time period > 6 months and <1 year (Group B) and 54 (29.7%) had been infected within a time period longer than 1 year from the examination date (Group C). Among the three groups, a positive T-cell reaction against the S antigen was reported in 47/58 (81%) of Group A, 61/69 (88.4%) of Group B and 40/54 (74.1%) of Group C (chi square, p = 0.27). T-cell reaction against the N antigen was present in 45/58 (77.6%) of Group A, 61/69 (88.4%) of Group B and 36/54 (66.7%) of Group C (chi square, p = 0.02). The median Spot-Forming Cells (SFC) count for the S antigen was 18 (range from 0-160) in Group A, 19 (range from 0-130) in Group B and 17 (range from 0-160) in Group C (Kruskal-Wallis test, p = 0.11; pairwise comparisons: groups A-B, p = 0.95; groups A-C, p = 0.89; groups B-C, p = 0.11). The median SFCs count for the N antigen was 14.5 (ranging from 0 to 116) for Group A, 24 (ranging from 0-168) in Group B and 16 (ranging from 0-112) for Group C (Kruskal-Wallis test, p = 0.01; pairwise comparisons: groups A-B, p = 0.02; groups A-C, p = 0.97; groups B-C, p = 0.03). CONCLUSIONS: Our data suggest that protective adaptive T-cellular immunity following natural infection by SARS-CoV-2 may persist for over 12 months, despite the undetectable humoral element.

Association of GSTM1 Polymorphism and Redox Potential with Idiopathic Male Infertility.

BACKGROUND: The aim of this case-control study is to investigate possible associations between GSTM1 polymorphism and redox potential with sperm parameters. METHODS: The study group consisted of sperm samples from 51 infertile men according to the WHO guidelines. The control group included 39 samples from men with normal seminal parameters. DNA was extracted and genotyped for the detection of the GSTM1 polymorphism. An evaluation of the static redox potential (sORP) using the MiOXSYSTM system was conducted. RESULTS: The frequency of the GSTM1-null genotype was higher in infertile male individuals (60.78%) than in the controls (41.03%) and was associated with a 2.228-fold increased risk for male infertility. Fertile controls carrying the GSTM1-null genotype presented a lower percentage of typical sperm morphology and lower slow progressive motility. An excess of redox potential was observed in infertile males compared to fertile ones. In the control group higher sORP values had a positive correlation with immotility percentage and a negative correlation regarding total motility. In the study group sORP values had a negative correlation with total count, concentration, and slow progressive motility. CONCLUSIONS: The present study highlights that GSTM1 polymorphism and redox potential affect both fertile and in fertile males. Moreover, redox potential levels could be used as an additional indicator along with the routine semen analysis for a comprehensive screening between infertile and fertile men.

ANRIL rs4977574 Gene Polymorphism in Women with Recurrent Pregnancy Loss.

BACKGROUND: ANRIL rs4977574 gene polymorphism has been associated with arterial thrombosis and cardiovascular disease development. ANRIL rs4977574 gene polymorphism could also be associated with recurrent pregnancy loss (RPL) since there is increasing evidence in favor of a potential shared pathophysiological mechanism with cardiovascular disease, potentially through arterial thrombosis. This study's goal is to investigate the differences in ANRIL rs4977574 gene polymorphism between women with and without RPL, if any, as well as a potential association with the number of pregnancy losses. METHODS: DNA was isolated from peripheral blood samples, and the sequence containing the polymorphism of interest was amplified with PCR. Results were visualized under UV light following electrophoresis in 3% agarose gel with ethidium bromide. ANRIL rs4977574 (A>G) prevalence was compared between 56 women with and 69 without RPL. Results were adjusted for women's age and BMI, while a stratified analysis was performed according to number of pregnancy losses. RESULTS: Allele A was significantly more prevalent in the control group compared to RPL women [31 (44.9%) vs. 14 (25%), p = 0.021]. Although not reaching statistical significance, a gradually decreasing prevalence of allele A with an increasing number of pregnancy losses was observed [31 (44.9%) in control, eight (30.7%) with two, six (23.1%) with three, and 0 (0.0%) with four pregnancy losses, p = 0.078]. Results were also similar following adjustment. CONCLUSIONS: This is the first study that demonstrates an association between RPL presence and ANRIL rs4977574 gene polymorphism (lower prevalence of allele A), while a difference according to the number of pregnancy losses cannot be excluded.

SARS-CoV-2 T Cell Immunity Responses following Natural Infection and Vaccination.

(1) Background: SARS-CoV-2 T cell immunity is rapidly activated following SARS-CoV-2 infection and vaccination and is crucial for controlling infection progression and severity. The aim of the present study was to compare the levels of T cell responses to SARS-CoV-2 between cohorts of subjects with hybrid immunity (convalescent and vaccinated), vaccinated naïve (non-exposed) and convalescent unvaccinated subjects. (2) Methods: We performed a retrospective descriptive analysis of data collected from the medical records of adult individuals who were consecutively examined at a large, private Medical Center of Attica from September 2021 to September 2022 in order to be examined on their own initiative for SARS-CoV-2 T cell immunity response. They were divided into three groups: Group A: SARS-CoV-2 convalescent and vaccinated subjects; Group B: SARS-CoV-2 naïve vaccinated subjects; Group C: SARS-CoV-2 convalescent unvaccinated subjects. The SARS-CoV-2 T cell response was estimated against spike (S) and nucleocapsid (N) structural proteins by performing the methodology T-SPOT.COVID test. (3) Results: A total of 530 subjects were retrospectively included in the study, 252 females (47.5%) and 278 (52.5%) males ranging from 13 to 92 years old (mean 55.68 ± 17.0 years). Among them, 66 (12.5%) were included in Group A, 284 (53.6%) in Group B and 180 (34.0%) in Group C. Among the three groups, a reaction against S antigen was reported in 58/66 (87.8%) of Group A, 175/284 (61.6%) of Group B and 146/180 (81.1%) of Group C (chi-square, p < 0.001). Reaction against N antigen was present in 49/66 (74.2%) of Group A and in 140/180 (77.7%) of Group C (chi-square, p = 0.841). The median SFC count for S antigen was 24 (range from 0-218) in Group A, 12 (range from 0-275) in Group B and 18 (range from 0-160) in Group C (Kruskal-Wallis test, p < 0.001; pairwise comparisons: groups A-B, p < 0.001; groups A-C, p = 0.147; groups B-C, p < 0.001). The median SFCs count for N antigen was 13 (range 0-82) for Group A and 18 (range 0-168) for Group C (Kruskal-Wallis test, p = 0.27 for A-C groups). (4) Conclusions: Our findings suggest that natural cellular immunity, either alone or combined with vaccination, confers stronger and more durable protection compared to vaccine-induced cellular immunity.

Expression of ZEB1 in different forms of endometriosis: A pilot study.

OBJECTIVES: Epithelial-Mesenchymal Transition (EMT), a cellular process in which epithelial cells lose epithelial characteristics while acquire mesenchymal features, is believed to contribute to migration and invasion abilities of the endometriotic cells. Studies on gene expression of the transcription factor ZEB1, a crucial transcription factor of EMT, show that there is probably a modified expression in the endometriotic lesions. The aim of the study was to compare the expression levels of ZEB1 in types of endometriotic lesions with different biological behavior such as endometriomas and deep infiltrating endometriotic nodules. STUDY DESIGN: We have studied 19 patients with endometriosis and 8 patients with benign gynecological lesions without endometriosis. The endometriosis patient group included 9 women with only endometriotic cysts without deep infiltrating endometriotic lesion (DIE) and 10 women with DIE who had developed concurrent endometriotic cysts. The technique applied to investigate ZEB1 expression levels is Real-Time PCR. The results of the reaction were normalized by simultaneously investigating the expression of the house-keeping gene G6PD. RESULTS: Analysis of the samples showed underexpression of ZEB1 in the eutopic endometrium of women with only endometriotic cysts when compared to normal endometrium. A tendency of higher ZEB1 expression, without reaching significant difference, was found between the endometriotic cysts and their paired eutopic endometrium. In women with DIE, no significant difference was found between their eutopic and normal endometrium. No significant difference was found between the endometriomas and DIE lesions. ZEB1 shows different expression profile in the endometriotic cysts of women with and without DIE when the cyst is compared to their paired eutopic endometrium. CONCLUSIONS: It therefore appears that ZEB1 expression differs between different types of endometriosis. The expression levels of ZEB1 in the eutopic endometrium could affect the development of infiltrating lesions or not. However, the most important observation is the different ZEB1 expression profile of endometriomas between women with and without DIE. Although, they both share the same histologic characteristics, they show different ZEB1 expression indicating different pathogenetic mechanisms of endometriomas in cases with and without DIE. Therefore, future research on endometriosis should consider DIE and ovarian endometriosis as different diseases.

Association between TP53 Arg72Pro variant and recurrent pregnancy loss in the Greek population.

OBJECTIVES: The present case-control study investigates whether TP53 Arg72Pro variant (rs1042522) serves as a risk factor for recurrent pregnancy loss (RPL) in Greek women. METHODS: The study group consisted of 100 patients with at least two miscarriages of unexplained etiology, before the 24th week of gestation. The control group included 106 women with no pregnancy loss history. DNA was extracted and genotyped using specific primers for PCR amplification of the Arg72 and Pro72 alleles. Sanger sequencing was used for the discrimination between heterozygotes and homozygotes for Arg72Pro variant. RESULTS: This is the first study demonstrating the statistically significant higher frequency of TP53 Arg72Pro variant in Greek RPL women compared to controls (38% vs. 6.6%; OR=8.6682, 95% CI: 3.6446-20.6160; p<0.0001). GC genotype (Arg/Pro) and CC genotype (Pro/Pro) were statistically more common in RPL patients than in controls (16% vs. 1.9%; p=0.0027, and 22 vs. 4.7%; p=0.0008, respectively). C allele frequency was statistically significant higher in RPL group than in controls (30.0 vs. 5.7%; p<0.0001). According to the inheritance mode analysis, the model that best fit the data was the dominant model (OR=8.67, 95% CI=3.64-20.62; p<0.0001). CONCLUSIONS: The is the first study disclosing strong evidence that TP53 rs1042522 is significantly associated with a higher risk for recurrent pregnancy loss in Greek women following a dominant model, thus, serving as a genetic marker for identifying women at increased risk of recurrent miscarriages.

Association of Tumor Necrosis Factor-α -308G>A, -238G>A and -376G>A polymorphisms with recurrent pregnancy loss risk in the Greek population.

BACKGROUND: Promoter region SNPs in TNF-α have been studied in association with Recurrent Pregnancy Loss (RPL) occurrence in various populations. Among them, -238G > A, -308G > A and - 376G > A have been frequently investigated for their potential role in recurrent abortions. The aim of the present study is to evaluate the correlation among TNF-α 238, TNF-α 308 and TNF-α 376 polymorphisms and recurrent pregnancy loss risk in Greek women. METHODS: This study included 94 Caucasian women with at least two miscarriages of unexplained aetiology, before the 20th week of gestation. The control group consisted of 89 Caucasian women of proven fertility, with no history of pregnancy loss. DNA samples were subjected to PCR amplification using specific primers. Sanger sequencing was applied to investigate the presence of TNF-α 238, TNF-α 308, TNF-α 376 polymorphisms in all samples. RESULTS: The TNF-α 238 and TNF-α 308 variants were both detected in RPL and control groups (7.45% vs 4.49 and 45.16% vs 36.73%, respectively), but with no statistically significant association (p-value 0.396 and 0.374, respectively). The TNF-α 376 variant was not detected at all in both control and RPL groups. When TNF-α 238 and TNF-α 308 genotypes were combined no association with RPL was detected (p-value = 0.694). In subgroup analysis by parity, RPL patients carrying the A allele reported less previous births. CONCLUSIONS: This is the first study demonstrating TNF-α 238 and TNF-α 308 gene expression and the absence of TNF-α 376 variant in Greek women with RPL. However, no association emerged between each polymorphism studied and the occurrence of recurrent pregnancy loss. Accordingly, TNF-α -308G > A, -238G > A and -376G > A variants are not considered genetic markers for identifying women at increased risk of recurrent pregnancy loss in the Greek population.

Meta-analysis of GnRH-antagonists versus GnRH-agonists in poor responder protocols.

PURPOSE: Considering the insufficient evidence supporting an ideal protocol for poor responder management in IVF/ICSI cycles, the aim of the current meta-analysis was to compare GnRH-antagonist versus GnRH-agonist protocols in poor responders, evaluating effectiveness and safety. METHODS: Meta-analysis was conducted using Medcalc 16.8 version software. Standardized mean differences (SMD), odds ratios (OR), and the respective 95% confidence intervals (CI) were determined appropriately. The Cochran Q statistic and the I2 test were used to assess studies' heterogeneity. RESULTS: GnRH-agonists were shown to correlate with fewer cancelled IVF/ICSI cycles (p = 0.044, OR = 1.268 > 1, 95% CI 1.007, 1.598), a larger number of embryos transferred (p = 0.008, SMD = - 0.230, 95% CI - 0.400, - 0.0599), and more clinical pregnancies (p = 0.018, OR = 0.748 < 1, 95% CI 0.588, 0.952). However, GnRH-antagonists resulted in a significantly shorter duration of ovarian stimulation (p = 0.007, SMD = - 0.426. 95% CI - 0.736, - 0.115). The number of oocytes and mature oocytes retrieved in both protocols did not differ statistically (p = 0.216, SMD = - 0.130, 95% CI - 0.337, 0.0763 and p = 0.807, SMD = - 0.0203, 95% CI - 0.183, 0.142, respectively). Moreover, a high heterogeneity among studies was observed regarding duration of ovarian stimulation (I2 = 90.6%), number of oocytes (I2 = 82.83%)/mature oocytes retrieved (I2 = 70.39%), and embryos transferred (I2 = 72.83%). CONCLUSIONS: Based on the present meta-analysis, agonist protocols could be suggested as a first choice approach, in terms of effectiveness. Due to the high studies' heterogeneity, results should be considered with caution. Accordingly, larger cohort studies and meta-analyses like the present one will enhance the robustness of the emerging results to identify the ideal protocol for poor responders.

Pathology of BRCA1- and BRCA2-associated Breast Cancers: Known and Less Known Connections.

INTRODUCTION: BRCA1/BRCA2 mutation carriers indefinitely comprise a distinct group of patients with breast cancer (BC), with their tumors displaying specific pathologic characteristics. Although these connections are known, they are not fully elucidated. We therefore sought to investigate the clinicopathologic characteristics and overall survival of Greek patients with BC carrying BRCA1/BRCA2 mutations. PATIENTS AND METHODS: Greek patients with BC diagnosed between 1999 and 2016, fulfilling the National Comprehensive Cancer Network criteria for genetic testing, were analyzed for BRCA1/BRCA2 mutations by Sanger sequencing or by a 94-gene panel. Medical records and pathology reports were retrospectively reviewed to retrieve patient and tumor baseline characteristics. Potential associations with mutation status were assessed using the Fisher exact, Pearson χ2, and Mann-Whitney tests. RESULTS: Of 2096 selected patients with BC, we identified 297 (14.2%) BRCA1 and 88 (4.2%) BRCA2 carriers. The mean age at BC diagnosis was 40 and 42.6 years, respectively (P = .02). Tumor histologic subtypes in BRCA1 and BRCA2 carriers were predominantly ductal (79%) followed by medullary (10%), and ductal (72%) followed by lobular (15%), respectively. A significantly higher percentage of BRCA2 tumors were human epidermal growth factor receptor 2-positive, compared with BRCA1 tumors (21.7% vs. 5.8%; P < .001). Second primary cancer diagnosis was more frequent in BRCA1 compared with BRCA2 mutation carriers (36.2% vs. 10.7%; P < .001), whereas there was no difference in 15-year overall survival (hazard ratio, 0.92; 95% confidence interval, 0.48-1.83; P = .804) between the 2 groups. CONCLUSIONS: These data confirm established observations in the pathology of BRCA-related tumors and provide further insight on the association of rare histologic entities with mutations in these genes, which can be clinically beneficial.

One in three highly selected Greek patients with breast cancer carries a loss-of-function variant in a cancer susceptibility gene.

BACKGROUND: Gene panel testing has become the norm for assessing breast cancer (BC) susceptibility, but actual cancer risks conferred by genes included in panels are not established. Contrarily, deciphering the missing hereditability on BC, through identification of novel candidates, remains a challenge. We aimed to investigate the mutation prevalence and spectra in a highly selected cohort of Greek patients with BC, questioning an extensive number of genes, implicated in cancer predisposition and DNA repair, while calculating gene-specific BC risks that can ultimately lead to important associations. METHODS: To further discern BC susceptibility, a comprehensive 94-cancer gene panel was implemented in a cohort of 1382 Greek patients with BC, highly selected for strong family history and/or very young age (<35 years) at diagnosis, followed by BC risk calculation, based on a case-control analysis. RESULTS: Herein, 31.5% of patients tested carried pathogenic variants (PVs) in 28 known, suspected or candidate BC predisposition genes. In total, 24.8% of the patients carried BRCA1/2 loss-of-function variants. An additional 6.7% carried PVs in additional genes, the vast majority of which can be offered meaningful clinical changes. Significant association to BC predisposition was observed for ATM, PALB2, TP53, RAD51C and CHEK2 PVs. Primarily, compared with controls, RAD51C PVs and CHEK2 damaging missense variants were associated with high (ORs 6.19 (Exome Aggregation Consortium (ExAC)) and 12.6 (Fabulous Ladies Over Seventy (FLOSSIES)), p<0.01) and moderate BC risk (ORs 3.79 (ExAC) and 5.9 (FLOSSIES), p<0.01), respectively. CONCLUSION: Studying a large and unique cohort of highly selected patients with BC, deriving from a population with founder effects, provides important insight on distinct associations, pivotal for patient management.

Prevalence and founder effect of the BRCA1 p.(Val1833Met) variant in the Greek population, with further evidence for pathogenicity and risk modification.

PURPOSE: Multiple lines of evidence have suggested a likely causative role in breast/ovarian cancer (BrCa/OvCa) predisposition for the BRCA1 p.(Val1833Met) variant, predominantly found among Greek patients. Our aim was to study the variant's prevalence and founder effect on the Greek population, while providing additional data for its pathogenicity. METHODS: We genotyped 3531 BrCa/OvCa patients using Sanger and next generation sequencing, as well as 1558 healthy, age-matched females with real-time PCR. Carriers underwent haplotype analysis to determine a founder effect. A co-segregation analysis was applied to estimate the likelihood ratio for pathogenicity. RESULTS: In total, 27 BrCa/OvCa patients (0.77%; 27/3531) were found to carry the p.(Val1833Met) variant. No carriers were identified in the control group diagnosis. A common shared haplotype, spanning 2.76 Mb on chromosome 17 was demonstrated among carriers, establishing the founder effect. BRCA1, p.(Val1833Met) is possibly a disease-associated variant, supported by a likelihood ratio of 1.88, while a correlation to ovarian cancer is suspected. CONCLUSIONS: Altogether, BRCA1, p.(Val1833Met) variant is a Greek founder and is very likely to predispose for BrCa/OvCa. Therefore, such carriers should be counselled accordingly, with clinical recommendations supporting surveillance and risk-reduction strategies, while providing the option for targeted therapeutic interventions.

PALB2 c.2257C>T truncating variant is a Greek founder and is associated with high breast cancer risk.

PALB2 loss-of-function variants play an important role in breast, pancreatic and possibly, ovarian and gastric cancer susceptibility. Their frequency can be influenced by founder effects, already described in some populations. Herein, we have assessed the possible founder effect of PALB2 c.2257C>T (p.Arg753*) truncating variant among Greek breast cancer patients, while investigating possible correlations with cancer diagnoses. Following a lead deriving from a background study of highly selected Greek breast cancer patients, a total of 2496 breast and 697 ovarian cancer patients were directly genotyped for the PALB2 c.2257C>T truncating variant. Consequently, haplotype analysis was conducted on identified carriers, using seven microsatellite markers. The prevalence of the PALB2 variant was 0.24% (6/2496) and 0.14% (1/697) among breast and ovarian cases, respectively. Family history seems to be an important factor for the variant identification, although not reaching statistical significance. Microsatellite analysis on 12 carriers revealed a common shared haplotype, spanning a chromosomal region of ~1.2 Mb; the variant was possibly introduced in the Greek population ~1600 years ago. The variant confers high breast cancer risk, as illustrated by comparison with publicly available control groups. Genetic testing for PALB2, especially for the Greek founder c.2257C>T truncating variant, should be seriously considered in Greek breast cancer cases, since such findings could assist appropriate clinical management for the patients and their families.

CHEK2 c.1100delC allele is rarely identified in Greek breast cancer cases.

The CHEK2 gene encodes a protein kinase that plays a crucial role in maintenance of genomic integrity and the DNA repair mechanism. CHEK2 germline mutations are associated with increased risk of breast cancer and other malignancies. From a clinical perspective, the most significant mutation identified is the c.1100delC mutation, which is associated with an approximately 25% lifetime breast cancer risk. The distribution of this mutation shows wide geographical variation; it is more prevalent in the Northern European countries and less common, or even absent, in Southern Europe. In order to estimate the frequency of the CHEK2 c.1100delC mutation in Greek breast cancer patients, we genotyped 2,449 patients (2,408 females and 41 males), which was the largest series ever tested for c.1100delC. The mean age of female and male breast cancer diagnosis was 49 and 59 years, respectively. All patients had previously tested negative for the Greek BRCA1 founder and recurrent mutations. The CHEK2 c.1100delC mutation was detected in 0.16% (4 of 2,408) of females, all of whom were diagnosed with breast cancer before the age of 50 years. Only one c.1100delC carrier was reported with breast cancer family history. The present study indicates that the CHEK2 c.1100delC mutation does not contribute substantially to hereditary breast cancer in patients of Greek descent.

Detection of RUNX2 gene expression in cumulus cells in women undergoing controlled ovarian stimulation.

BACKGROUND: RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. METHODS: A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. RESULTS: Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0). Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52). From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013), higher number of retrieved oocytes (p = 0.016), higher basal LH serum levels (p = 0.016) and higher peak estradiol levels (p = 0.013), while the number of fertilized oocytes differed marginally between the two groups (p = 0.089). Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021) and E2 levels (OR = 0.25, p = 0.026). CONCLUSIONS: Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.

LH receptor gene expression in cumulus cells in women entering an ART program.

PURPOSE: Luteinizing hormone (LH) exerts its actions through its receptor (LHR), which is mainly expressed in theca cells and to a lesser extent in oocytes, granulosa and cumulus cells. The aim of the present study was the investigation of a possible correlation between LHR gene and LHR splice variants expression in cumulus cells and ovarian response as well as ART outcome. METHODS: Forty patients undergoing ICSI treatment for male factor infertility underwent a long luteal GnRH-agonist downregulation protocol with a fixed 5-day rLH pre-treatment prior to rFSH stimulation and samples of cumulus cells were collected on the day of egg collection. RNA extraction and cDNA preparation was followed by LHR gene expression investigation through real-time PCR. Furthermore, cumulus cells were investigated for the detection of LHR splice variants using reverse transcription PCR. RESULTS: Concerning LHR expression in cumulus cells, a statistically significant negative association was observed with the duration of ovarian stimulation (odds ratio = 0.23, p = 0.012). Interestingly, 6 over 7 women who fell pregnant expressed at least two specific types of LHR splice variants (735 bp, 621 bp), while only 1 out of 19 women that did not express any splice variant achieved a pregnancy. CONCLUSIONS: Consequently, the present study provide a step towards a new role of LHR gene expression profiling as a biomarker in the prediction of ovarian response at least in terms of duration of stimulation and also a tentative role of LHR splice variants expression in the prediction of pregnancy success.