Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Perioperative Blood Transfusions Are Associated with a Higher Incidence of Thromboembolic Events After TKA: An Analysis of 333,463 TKAs

BackgroundGiven the morbidity, mortality, and financial burden associated with venous thromboembolism (VTE) after TKA, orthopaedic providers continually seek to identify risk factors associated with this devastating complication. The association between perioperative transfusion status and VTE risk has not been thoroughly explored, with previous studies evaluating this relationship being limited in both generalizability and power.Questions/purposesTherefore, we sought to determine whether perioperative transfusions were associated with an increased risk of (1) pulmonary embolism (PE) or (2) deep vein thrombosis (DVT) after primary TKA in a large, multi-institutional sample.MethodsThe American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database was implemented for our analysis. The definitions of complications, such as DVT and PE, and risk adjustment validation is monitored by the central ACS NSQIP office to ensure participating hospitals are adhering to the same guidelines to log patients. Additionally, both preoperative and intraoperative/72 hour postoperative transfusion status is included for all patients. Therefore, ACS NSQIP was determined to be the most appropriate database for our analysis. All patients who underwent primary TKA between 2011 and 2018 were identified using Current Procedural Terminology code 27447. Primary TKAs designated as “non-elective” were excluded, thereby providing a cohort composed solely of patients undergoing unilateral primary elective TKA for further analysis. The final analysis included 333,463 patients undergoing TKA (mean age 67 ± 9 years, 62% female). Preoperative transfusions were received by < 0.01% (48 of 333,463) of the patients, while 4% (14,590 of 333,463) received a transfusion within the interim between the start of surgery up to 72 hours postoperatively. All missing values were imputed through multiple imputation by chained equation to avoid variable availability-based selection and the subsequent listwise deletion-associated bias in the estimate of parameters. A multivariable logistic regression analysis was conducted using variables identified in a univariate model to calculate adjusted odds ratios and 95% confidence intervals for risk factors associated with symptomatic DVT and/or PE. For variables that maintained significance in the multivariable model, an additional model without confounders was used to generate fully adjusted ORs and 95% CIs. A propensity score matched comparison between recipients versus nonrecipients (1:1) of transfusion (preoperative and intraoperative/72 hours postoperative) was then conducted to evaluate the independent association between DVT/PE development and patients’ transfusion status. Significance was determined at a p value < 0.05.ResultsAdjusted multivariable regression analysis accounting for patient age, sex, race, BMI, American Society of Anesthesiologists (ASA) class and baseline comorbidities demonstrated the absence of an association between preoperative (OR 1.75 [95% CI 0.24 to 12.7]; p = 0.58) or intraoperative/72 hours postoperative (OR 1.12 [95% CI 0.93 to 1.35]; p = 0.23) transfusions and higher odds of developing PE. Similar findings were demonstrated after propensity score matching. Although multivariable regression demonstrated the absence of an association between preoperative transfusion and the odds of developing DVT within the 30-day postoperative period (OR 1.85 [95% CI 0.43 to 8.05]; p = 0.41), intraoperative/postoperative transfusion was associated with higher odds of DVT development (OR 3.68 [95% CI 1.14 to 1.53]; p < 0.001) relative to transfusion naïve patients. However, this significance was lost after propensity score matching.ConclusionAfter controlling for various potential confounding variables such as ASA Class, age, anesthesia type, and BMI, the receipt of an intra- or postoperative transfusion was found to be associated with an increased risk of DVT. Our findings should encourage orthopaedic providers to strictly adhere to blood management protocols, further tighten transfusion eligibility, and adjust surgical approach and implant type to reduce the incidence of transfusion among patients with other DVT risk factors. Additionally, our findings should encourage a multidisciplinary approach to VTE prophylaxis and prevention, as well as to blood transfusion guideline adherence, among all providers of the care team.Level of EvidenceLevel III, therapeutic study.     

Inclusion complexation of furosemide in cyclodextrins. Part 2: Implication on bioavailability

The effect of inclusion complexation of furosemide in cyclodextrins (CyD) on the bioavailability of the drug was studied on normal human volunteers. The excretion rate of the drug was determined by HPLC for a period of 24 h. post dosing. Several pharmacokinetic parameters were calculated and statistically analyzed. viz., peak excretion rate, peak excretion time, half peak time, elimination rate constant, area under the excretion rate time-curve as well as the total amount of drug excreted. The obtained results show that on administration of furosemide in the form of an inclusion complex in CyDs particularly beta-CyD or its dimethyl derivative may improve its biological performance, taking into consideration that furosemide is characterized by a rapid onset and a short duration of action. Inclusion complexation of furosemide in CyDs leads to a more or less delay in its onset of action, a significant increase in its duration of action as well as significant augmentation in its overall biological availability.     

A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma

We examined the relationship between a functional polymorphism (667C-- >T, ala-->val) of the methylenetetrahydrofolate reductase gene (MTHFR) and the risk of colorectal adenomas in the prospective Nurses' Health Study. Among 257 incident polyp cases and 713 controls, the MTHFR val/val polymorphism [relative risk (RR) = 1.35, 95% confidence interval (CI) 0.84-2.17] was not significantly associated with risk of adenomas. This lack of association was observed for both small (RR = 1.36, 95% CI 0.76-2.45) and large (RR = 1.32, 95% CI 0.66-2.66) adenomas. Furthermore, there was no significant interaction between this polymorphism and consumption of either folate, methionine or alcohol. We also examined the relationship of a newly identified polymorphism (asp919gly) of the methionine synthase gene (MS) with the risk of colorectal adenomas in the same population. The MS gly/gly polymorphism was also not significantly associated with risk of colorectal adenomas (RR = 0.66, 95% CI 0.26-1.70). These results, which need to be confirmed in other studies, suggest that the MTHFR val/val polymorphism, which has been previously inversely associated with risk of colorectal cancer, plays a role only in a late stage (adenoma-- >carcinoma) of colorectal tumorigenesis, and/or may protect against malignant transformation in the subset of benign adenomas, which may progress to malignancy.