Evaluation of Oxidative and Nitrosative Stress Markers Related To Inflammation in The Cumulus Cells and Follicular Fluid of Women Undergoing Intracytoplasmic Sperm Injection: A Prospective Study.

BACKGROUND: Oxidative/nitrosative stress in the oocyte microenvironment could have an impact on intracytoplasmic sperm injection (ICSI) outcomes. The presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can stimulate the secretion of pro-inflammatory cytokines, leading to chronic inflammation and potentially affecting embryo as well as oocyte quality. This study aimed to examine the relationship of lipid peroxidation [measured by the malondialdehyde (MDA) assay] with protein carbonyl [measured by the 2,4 dinitrophenylhydrazine (DNPH) assay] levels in cumulus cells (CCs), as well as nitric oxide (NO), peroxynitrite (ONOO-), and C-reactive protein (CRP) levels in follicular fluid (FF). The potential relationship of these levels with ICSI outcome was also evaluated. MATERIALS AND METHODS: In this prospective study, 63 FF samples and their corresponding CCs were collected for ICSI procedures. Spectrophotometry was used to assess levels of DNPH, MDA, NO, and ONOO-. CRP levels were evaluated using an immunoturbidimetric assay. RESULTS: The patients under 37 years with normal ovarian reserve had significantly lower levels of MDA, DNPH, NO, ONOO-, and CRP compared to those over 37 years. Additionally, we observed higher levels of MDA, DNPH, NO, ONOO-, and CRP in the group with an oocyte maturity rate of less than 60%. No significant difference was observed between the DNPH levels and factors such as infertility duration, embryo quality, pregnancy, or the number of retrieved oocytes. A higher level of MDA, NO, ONOO-, and CRP was found to be significantly related to the lower number of retrieved oocytes, longer periods of infertility, poor embryo quality, and negative pregnancy outcomes. CONCLUSION: Oxidative/nitrosative stress, linking to inflammation in the oocyte microenvironment, can be considered as a potentially useful biomarker for assessing the development and competence of oocytes and embryos and predicting ICSI outcomes.

Epidemiological, Clinicopathological and Prognosis Features of Moroccan Patients with Nasopharyngeal Carcinoma.

PROPOSAL: A distinct epidemiology, etiology, clinical characteristics, and therapeutic outcomes characterize nasopharyngeal carcinoma (NPC) from other head and neck cancers. An actualized analysis of NPC patients' features enables a global view of NPC management. Accordingly, the current study investigated the epidemiological and clinical characteristics of Moroccan patients with NPC, as well as their 4-years survival outcomes and influencing prognostic factors. METHODS: We prospectively analyzed data of 142 histologically confirmed Moroccan patients with NPC between October 2016 and February 2019. Kaplan-Meier and Cox regression analyses were used to assess predictive prognostic factors related to NPC. All analyses were conducted using SPSS version 21 statistical software. RESULTS: In the present study, a net male predominance was found, with a mean age of 44±16.3 years old. Advanced stages of NPC were observed in 64.1% of patients, and 32.4% of patients presented with distant metastasis at diagnosis. The 4-years overall survival, locoregional relapse-free survival, distant metastasis-free survival and progression-free survival were 68.0%, 63.0%, 53.9%, and 39.9%, respectively. Age, N category and distant metastasis were identified as the most important independent prognosis factors for NPC in this cohort (p<0.05). CONCLUSION: In conclusion, NPC affects young adults and is frequently diagnosed at advanced disease stages, impacting therefore negatively patients survival; which is in line with data from endemic areas for NPC. The current study clearly highlights that a greater attention should be directed to improving the management of this aggressive malignancy.

Assessment of cell-free DNA and apoptosis in an oocyte microenvironment: promising biomarkers to predict intracytoplasmic sperm injection outcomes.

Cell-free DNA (cf-DNA) is defined as DNA fragments that are released into the body fluids from apoptosis or necrosis cells, including follicular fluid (FF), which can affect the microenvironment of the oocyte associated with infertility. We aimed to investigate a relationship between apoptosis of cumulus cells (CCs) and cf-DNA levels in FF and clinical outcomes of women undergoing intracytoplasmic sperm injection (ICSI). Therefore, 82 FF samples were collected, and the corresponding CCs were isolated for ICSI procedures. FF cf-DNA concentration was quantified using ALU-quantitative polymerase chain reaction (PCR), and CCs DNA fragmentation index (DFI) was evaluated by the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) method. We found that cf-DNA and DFI levels were significantly higher in FF and CCs samples related to the age of women ≥37 years compared with the age of women < 37 years. Moreover, in older and younger women, FF cf-DNA and CCs DFI levels were significantly lower when the anti-Müllerian hormone (AMH) level was > 1.1 ng/ml compared with when AMH ≤ 1.1 ng/ml. In addition, patients with a low number of retrieved oocytes ≤ 6 had significantly higher levels of CCs DFI and FF cf-DNA than women with a higher number of retrieved oocytes > 6. Additionally, we observed that higher levels of cf-DNA and DFI were associated with poor oocyte maturity and poor embryo quality. Finally, cf-DNA and DFI levels were significantly lower in pregnant women than in non-pregnant ones. We conclude that DFI and cf-DNA levels in the oocyte microenvironment could have potential use in evaluating oocyte and embryo developmental competence.

Comparison between density gradient centrifugation method, an extended version of the horizontal swim up method and the combination of both for sperm selection.

OBJECTIVE: To compare the degree of efficiency between density gradient centrifugation (DGC) method and an extended horizontal swim-up (SU) method. METHODS: A total of 97 couples undergoing in vitro fertilization were enrolled in the study. Semen samples were divided into three aliquots and treated using DGC, extended horizontal SU, and combined methods. DNA fragmentation and chromatin decondensation were detected in native semen samples and their three corresponding aliquots. The corresponding mature oocytes of each semen sample were divided into two sibling cultures. The first sibling culture was microinjected with semen pellets from DGC, and the second sibling culture was microinjected with semen pellets from the combination of both methods. Fertilization rate and embryonic development were assessed at day 3. RESULTS: DNA fragmentation and chromatin decondensation was significantly low in DGC and extended horizontal SU samples; however, the rates of DNA fragmentation and chromatin decondensation were significantly lower in extended horizontal SU samples than in DGC samples. The lowest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with both methods. The highest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with DGC. No significant difference was found in the fertilization rate or day 3 embryos between sibling cultures. CONCLUSION: The combination of DGC and the extended horizontal SU techniques is best for giving the lowest rates of sperm DNA fragmentation and chromatin decondensation.

Neutralization Capacity of Tissue Alterations Caused by the Venoms of the Most Dangerous Scorpions in North Africa Using a Selective Antivenom.

In North Africa, scorpion stings pose an urgent public health problem, particularly for children with high morbidity and mortality rates. The main species implicated are the Androctonus mauretanicus (Am), Androctonus australis hector (Aah), and Buthus occitanus (Bo). Immunotherapy is the specific therapeutic approach aimed at directly neutralizing toxins, despite their severity and rapid diffusion. In the present study, we evaluate, histologically and immunohistologically, the neutralization potency of the selective antivenom produced against, among other species, the Am, Aah, and Bo at the level of the tissue alterations in Swiss mice, as experimental subjects. Firstly, the lethal doses 50 test was conducted to assess the venom's toxic activity, and then the median effective dose of the antivenom was determined against each venom. The histological and immunohistological analyses were performed by injecting the sublethal dose of venom, the complex venom and antivenom, or the antivenom 2 h following inoculation of venom. Our study revealed the highest toxicity of the Am, followed by the Aah and then the Bo venom. The neutralizing ability and effectiveness of the antivenom to completely or partially neutralize the tissular damages were demonstrated in all organs studied: brain, heart, lungs, liver, and kidneys. Our results highlighted the important cytoplasmic and membranous staining in the heart compared to the brain tissue for the three scorpion venoms. Therefore, the scorpionic antivenoms are able to reach their target even at the tissue level. Immunotherapy represents the specific and recommended treatment against the scorpionic stings in North Africa.

Pretreatment [18F]FDG PET/CT and MRI in the prognosis of nasopharyngeal carcinoma.

OBJECTIVE: The present study aimed to assess the prognostic interest of metabolic and anatomic parameters derived from 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography ([18F]FDG PET/CT) and head and neck magnetic resonance imaging (HN-MRI) for better management of nasopharyngeal carcinoma (NPC). METHODS: In this study, pre-treatment [18F]FDG PET/CT and HN-MRI parameters of NPC patients diagnosed between January 2017 and December 2018, were prospectively investigated. Correlation between those parameters and 4-year patient's survival outcomes was evaluated using Kaplan-Meier and Cox-regression analyses. RESULTS: Our results revealed a significant association between pre-treatment nodal-maximum standardized uptake value (N-SUV max) and N categories (p = 0.01), between pre-treatment node-to-tumor SUV ratio (NTR) and both tumor size (p = 0.01) and N categories (p = 0.009), as well as between metabolic tumor volume (MTV) and both tumor size and NPC overall stage (p < 0.000). In multivariate analyses, pre-treatment N-SUV max, NTR and MTV were significant independent predictors of overall survival, distant metastasis-free survival, and progression-free survival (PFS) (p < 0.05). N-SUV max and MTV were also found to be significant independent predictors of loco-regional recurrence-free survival (p < 0.05), whereas HN-MRI detection of skull-base bone invasion was an independent factor associated with worse PFS in NPC (p = 0.03). CONCLUSIONS: The present study highlights N-SUV max, NTR and MTV derived from [18F]FDG PET/CT, and skull-base bone invasion defined by HN-MRI, as promising metabolic and anatomic prognosis biomarkers for NPC.

Anti-Cancer Activity of Buthus occitanus Venom on Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most dominant primary liver cancer, which can be caused by chronic hepatitis virus infections and other environmental factors. Resection, liver transplantation, and local ablation are only a few of the highly effective and curative procedures presently accessible. However, other complementary treatments can reduce cancer treatment side effects. In this present work, we evaluated the activity of Moroccan scorpion venom Buthus occitanus and its fractions obtained by chromatography gel filtration against HCC cells using a 3D cell culture model. The venom was fractionated by gel filtration chromatography, each fraction and the crude venom was tested on normal hepatocytes (Fa2N-4 cells). Additionally, the fractions and the crude venom were tested on MCTSs (multicellular tumor spheroids), and this latter was generated by cultivate Huh7.5 cancer cell line with WI38 cells, LX2 cells, and human endothelial cells (HUVEC). Our results indicate that Buthus occitanus venom toxin has no cytotoxic effects on normal hepatocytes. Moreover, it is reported that F3 fraction could significantly inhibit the MCTS cells. Other Protein Separation Techniques (High-performance liquid chromatography) are needed in order to identify the most active molecule.

Prognostic value of Epstein-Barr virus DNA load in nasopharyngeal carcinoma: a meta-analysis.

The present meta-analysis was conducted to evaluate the prognostic value of pre and post-Epstein Barr Virus (EBV) DNA load testing and to assess the clinical benefit of using this molecular approach in the prognosis for a better nasopharyngeal carcinoma (NPC) management. Relevant studies were searched in different database until May 2020. Patient´s outcomes overall survival (OS), disease free survival (DFS), progression-free survival (PFS), distant-metastasis-free survival (DMFS), and local-regional-failure-free survival (LRFS), hazard ratios (HRs) and 95% confidence intervals (CIs) were extracted from selected studies. The association of pre and post-EBV DNA load and survival outcomes was assessed using review manager and the pooled HRs with 95% CIs were calculated. Twenty-six eligible studies were included in this meta-analysis, with a total of 9966 patients. Pooled HRs showed that EBV DNA levels before and after treatment are significantly associated with survival outcomes, with HR (95% CI) of 2.09 [1.74, 2.51] for OS, 1.77 [1.19, 2.62] for DFS, 2.53 [2.18, 2.92] for DMFS, 1.78 [1.45, 2.19] for LRFS and 2.17 [1.91, 2.47] for PFS in pre-EBV DNA, and an HR (95%) of 4.52 [2.44, 8.36], 4.08 [2.38, 6.99], 5.59 [ 3.58, 8.71] and 8.88 [5.29, 14.90] for OS, DFS and PFS and DMFS in post-EBV DNA, respectively. High pre and post-EBV DNA levels were significantly associated with poor NPC patient´s survival outcomes; which clearly confirm the high interest to introduce viral EBV DNA load as a prognostic biomarker for NPC management.

Induced pathophysiological alterations by the venoms of the most dangerous Moroccan scorpions Androctonus mauretanicus and Buthus occitanus: A comparative pathophysiological and toxic-symptoms study.

Scorpion envenomation is a serious public health issue. Androctonus mauretanicus (Am) and Buthus occitanus (Bo) are the most dangerous scorpions in Morocco. Despite their medical relevance, no study has yet related their kinetics of symptom apparition and the consequent tissue disorders at the same interval post-injection. This work achieved the first comparative pathophysiological and toxic-symptoms study between the Am and Bo venoms from a biochemical, toxicological and physiopathological standpoint. The activity of venoms and their subletal dose were determined by administration of increasing concentrations of the venoms. 30, 60 and 120 min following the experimental envenomation in mice, the profile of clinical symptoms was underlined and the main organs: brain, heart, lungs, liver and kidneys were removed for histological examination. The Am venom is a rich source of proteins and three-times more toxic than the Bo. The most observed clinical symptoms are neurological and cardiopulmonary. The Am venom caused histopathological alterations at 30, 60, and 120 min which were more important than the Bo. This study highlighted that both venoms exhibited a strong toxicity with variable intensities. Moreover, we showed the presence of correlation between the level of histopathological disorders observed and the intensity of signs appeared at the same time following venom inoculation.

The Dynamic Change in Plasma Epstein-Barr Virus DNA Load over a Long-Term Follow-Up Period Predicts Prognosis in Nasopharyngeal Carcinoma.

The current study was designed to investigate the changes in the circulating Epstein−Barr virus DNA load (EBV DNA) at various time points before and after treatment and its clinical significance in nasopharyngeal carcinoma (NPC). A total of 142 patients with NPC were prospectively enrolled in this study. The plasma EBV DNA concentration was measured before and after treatment using qPCR. The prognostic values of the EBV DNA load were analyzed using the Kaplan−Meier and Cox regression tests. Following multivariate analysis, our data showed that high pre-EBV DNA loads were associated with significantly poorer distant metastasis free survival (DMFS) and progression free survival (PFS); detectable end-EBV DNA loads were associated with significantly worse loco-regional recurrence free survival (LRRFS) and PFS, and the detecTable 6 months-post-EBV DNA loads were associated with significantly poorer overall survival (OS), DMFS and PFS (p < 0.05). Additionally, combining the pre-EBV DNA load and the stage of the disease, our results showed that patients at stage III-IVA with a low pre-EBV DNA load had similar survival rates as patients at stage II with a low or high pre-EBV DNA load, but had better survival rates than those at stage III-IVA with a high pre-EBV DNA load. Taken together, we showed that the change of the EBV DNA load measured at several time points was more valuable than at any single time point for predicting patients' survival for NPC. Furthermore, combining the pre-EBV DNA load and the TNM classification could help to formulate an improved prognostic model for this cancer.

Anti-Cancer Effect of Moroccan Cobra Naja haje Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

Mass spectrometry-based top-down and bottom-up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom.

Buthus occitanus (B. occitanus) is one of the most dangerous scorpions in the world. Despite the involvement of B. occitanus scorpion in severe cases of envenomation in Morocco, no study has focused yet on the proteomic composition of the Moroccan B. occitanus scorpion venom. Mass spectrometry-based proteomic techniques are commonly used in the study of scorpion venoms. The implementation of top-down and bottom-up approaches for proteomic analyses facilitates screening by allowing a global view of the structural aspects of such complex matrices. Here, we provide a partial overview of the venom of B. occitanus scorpion, in order to explore the diversity of its toxins and hereafter understand their effects. To this end, a combination of top-down and bottom-up approaches was applied using nano-high liquid chromatography coupled to nano-electrospray tandem mass spectrometry (nano-LC-ESI MS/MS). The LC-MS results showed that B. occitanus venom contains around 200 molecular masses ranging from 1868 to 16 720 Da, the most representative of which are those between 5000 and 8000 Da. Interestingly, combined top-down and bottom-up LC-MS/MS results allowed the identification of several toxins, which were mainly those acting on ion channels, including those targeting sodium (NaScTxs), potassium (KScTxs), chloride (ClScTxs), and calcium channels (CaScTx), as well as antimicrobial peptides (AMPs), amphipathic peptides, myotropic neuropeptides, and hypothetical secreted proteins. This study reveals the molecular diversity of B. occitanus scorpion venom and identifies components that may have useful pharmacological activities.

Circulating cell-free epstein-barr virus DNA levels and clinical features in Moroccan patients with nasopharyngeal carcinoma.

BACKGROUND: The identification of effective prognosis biomarkers for nasopharyngeal carcinoma (NPC) is crucial to improve treatment and patient outcomes. In the present study, we have attempted to evaluate the correlation between pre-treatment plasmatic Epstein-Barr virus (EBV) DNA load and the conventional prognostic factors in Moroccan patients with NPC. METHODS: The present study was conducted on 121 histologically confirmed NPC patients, recruited from January 2017 to December 2018. Circulating levels of EBV DNA were measured before therapy initiation using real-time quantitative PCR. RESULTS: Overall, undifferentiated non-keratinizingcarcinoma type was the most common histological type (90.1 %), and 61.8 % of patients were diagnosed at an advanced disease stage (IV). Results of pre-treatment plasma EBV load showed that 90.9 % of patients had detectable EBV DNA, with a median plasmatic viral load of 7710 IU/ml. The correlation between pre-treatment EBV DNA load and the conventional prognostic factors showed a significant association with patients' age (p = 0.01), tumor classification (p = 0.01), lymph node status (p = 0.003), metastasis status (p = 0.00) and overall cancer stage (p = 0.01). Unexpectedly, a significant higher level of pre-treatment EBV DNA was also found in plasma of NPC patients with a family history of cancer (p = 0.04). The risk of NPC mortality in patients with high pretreatment EBVDNA levels was significantly higher than that of those with low pre-treatment plasma EBV-DNA levels (p < 0.05). Furthermore, patients with high pre-treatment EBV-DNA levels (≥ 2000, ≥ 4000) had a significant low overall survival (OS) rates (p < 0.05). Interestingly, lymph node involvement, metastasis status and OS were found to be the most important factors influencing the EBV DNA load in NPC patients. CONCLUSIONS: The results of the present study clearly showed a high association between pre-treatment EBV DNA load, the crucial classical prognostic factors (T, N, M and disease stage) of NPC and OS, suggesting that pre-treatment EBV DNA can be a useful prognostic biomarker in clinical decision-making and improving NPC treatment in Morocco.

[Current antiplatelet agents, new inhibitors and therapeutic targets]. / Antiplaquettaires actuels, en cours de développement et cibles thérapeutiques.

Cardiovascular diseases are the leading cause of deaths in the world. Platelets play a major role in the occurrence of these diseases and the development of antiplatelet drugs is a priority in the fight against cardiovascular diseases-associated mortality. Aspirin and thienopyridine-based P2Y12 inhibitors are the main drugs currently used. These molecules target the initiation of platelets activation and are responsible for a moderate inhibitory action. Other antiplatelet agents, as glycoprotein (GP) IIb/IIIa antagonists, inhibit platelet aggregation independently of initial activation-associated pathways, but are responsible for increased hemorrhagic events. Regarding each antiplatelet agent's specific characteristics, the prescription of these drugs must take into account the type of cardiovascular event, the age of the patient, the past medical history, and the potential hemorrhagic adverse events. Thus, there is a need for the development of new molecules with a more targeted effect, maintaining optimal efficiency but with a reduction of the hemorrhagic risk, which is the principal limitation of these treatments.

TITLE: Antiplaquettaires actuels, en cours de développement et cibles thérapeutiques. ABSTRACT: Les maladies cardiovasculaires (MCV) sont la première cause de mortalité dans le monde. Les plaquettes jouent un rôle majeur dans le développement de ces maladies et la mise au point d'antiplaquettaires efficaces constitue une priorité dans le cadre de la lutte contre la mortalité liée aux MCV. L'aspirine et les médicaments de la famille des thiénopyridines sont les agents antiplaquettaires les plus utilisés actuellement. Ces médicaments ciblent des voies de signalisation impliquées dans l'initiation de l'agrégation, exerçant ainsi un effet antiplaquettaire modéré. D'autres médicaments aux effets plus importants, comme les molécules dirigées contre le récepteur GPIIb/IIIa, inhibent l'agrégation plaquettaire indépendamment de la voie de signalisation initiant l'activation plaquettaire, mais ils sont associés à des complications hémorragiques majorées. Étant données les caractéristiques spécifiques de chacun de ces agents antiplaquettaires, leur prescription nécessite de prendre en compte le type d'évènement cardio-vasculaire, l'âge et les comorbidités du patient traité et, bien sûr, les effets secondaires hémorragiques potentiels de la molécule qui est prescrite. Apparaît donc la nécessité de mettre au point de nouvelles molécules ayant un effet plus ciblé, gardant une efficacité optimale, mais permettant une réduction du risque hémorragique qui constitue la principale limite des médicaments antiplaquettaires.

Chromosomal Abnormalities in Patients with Intellectual Disability: A 21-Year Retrospective Study.

BACKGROUND: Intellectual disability (ID) has been defined as a considerably reduced ability to understand new or complex information and to learn new skills. It is associated with life-long intellectual and adaptive functioning impairments that have a profound impact on individuals, families, and society. It affects about 3% of the general population. ID often comes out with other mental conditions like attention deficit, hyperactivity, and autism spectrum disorders (ASD), and it can be part of a malformation syndrome that affects other organs. It may be syndromic (S-ID) or non-syndromic (NS-ID). OBJECTIVE: The aims of this study were to identify the profile of intellectually disable patients being referred for cytogenetic analysis in Morocco, to determine the prevalence of chromosomal abnormalities in a Moroccan group, and to compare the results with those of analogous studies from other countries. PARTICIPANTS: We included data from Moroccan patients with NS-ID and others with S-ID (mostly Down syndrome cases) who have been referred between 1996 and 2016. 1,626 patients were involved in this study, 1,200 were referred with a clinical diagnosis of Down syndrome, 37 were clinically diagnosed for ASD with ID, and 389 were suspected of NS-ID. RESULTS: We identified 1,200 cases of Down syndrome. In 1,096 analyses (91.3%), a cytogenetic variant of trisomy 21 was identified: standard trisomy 21 in 1,037 cases (94.6%), a translocation in 34 cases (3.10%), and mosaicism in 25 cases (2.3%). The cytogenetic analysis among ASD with ID cases did not reveal any specific chromosomal abnormalities. The present study also shows that chromosomal abnormalities were present in 6.43% of the patients with NS-ID (25 abnormal karyotypes out of 389 NS-ID cases). Autosomal structural abnormalities were the largest proportion of chromosomal aberrations. CONCLUSION: The high rate of chromosomal abnormalities found in the Moroccan patients studied demonstrates the capital importance of cytogenetic evaluation in patients who show ID or any clinical development abnormality.