The effects of case/control ratio and sample size on genome-wide association studies: A simulation study.

BACKGROUND: Genome-wide association studies (GWAS) is a useful tool for the detection of disease or quantitative trait-related genetic variations in the veterinary field. For a binary trait, a case/control experiment is designed in GWAS. However, there is limited information on the optimal case/control and sample size in GWAS. OBJECTIVES: In this study, it was aimed to detect the effects of case/control ratio and sample size for GWAS using computer simulation under certain assumptions. METHOD: Using the PLINK software, we simulated three different disease scenarios. In scenario 1, we simulated 10 different case/control ratios with increasing ratio of cases to controls. In scenario 2, we did versa of scenario 1 with the increasing ratio of controls to cases. In scenarios 1 and 2, sample size gradually was increased with the change case/control ratios. In scenario 3, the total sample size was fixed to 2000 to see real effects of case/control ratio on the number of disease-related single nucleotide polymorphisms (SNPs). RESULTS: The results showed that the number of disease-related SNPs were the highest when the case/control ratio is close to 1:1 in scenarios 1 and 2 and did not change with an increase in sample size. Similarly, the number of disease-related SNPs was the highest in case/control ratios 1:1 in scenario 3. However, unbalanced case/control ratio caused the detection of lower number of disease-related SNPs in scenario 3. The estimated average power of SNPs was highest when case/control ratio is 1:1 in all scenarios. CONCLUSIONS: All findings led to the conclusion that an increase in sample size may enhance the statistical power of GWAS when the number of cases is small. In addition, case/control ratio 1:1 may be the optimal ratio for GWAS. These findings may be valuable not only for veterinary field but also for human clinical experiments.

Anti-Müllerian hormone: A novel biomarker for detecting bovine freemartinism.

The anti-Müllerian hormone (AMH) indicates ovarian reserve in cattle, maintaining a consistent trajectory post-puberty. In heterosexual pregnancies, the development of the Müllerian duct in female foetuses is inhibited, resulting in an anticipated minimal or absent ovarian reserve capacity. This investigation aimed to compare AMH levels in healthy Holstein heifers that had reached puberty with those of freemartin animals of the same breed and age. The study incorporated Holstein heifers reaching puberty between 11 and 15 months of age in Group 1 (G1, n = 20) and freemartin animals in Group 2 (G2, n = 19, 16). AMH measurements (AMH-1/AMH-2) were recorded at 12-day intervals for the study participants. Notably, AMH levels in three freemartin animals could not be detected, prompting statistical analysis based on measurements from the remaining 16 freemartin animals in G2. A statistically significant correlation was observed between two separate measurements in G1 and G2 (p < .001). Furthermore, AMH-1 and AMH-2 levels were statistically higher in G1 than in G2 (p < .001). In G1, AMH-1 levels ranged from 227 to 677 pg/mL, with an average of 367.3 ± 25.5 pg/mL, and AMH-2 levels ranged from 234 to 645 pg/mL, with an average of 380.8 ± 24.4 pg/mL. Conversely, in G2, AMH-1 levels ranged from 10 to 72 pg/mL, with an average of 26.8 ± 4.44 pg/mL, and AMH-2 levels ranged from 12 to 68 pg/mL, with an average of 28.75 ± 4.18 pg/mL. The mean AMH levels in G1 were approximately 14 times higher than in G2 (p < .001). Consequently, ROC analysis utilizing AMH-1 and AMH-2 data established cut-off values of ≤72 and ≤ 68 pg/mL respectively for distinguishing freemartin animals. In conclusion, AMH could be used as a reliable biomarker for identifying Holstein freemartin animals.

Expression and localization of insulin-like growth factor gene family members in the caprine ovarian and uterine tissues during different pregnancy stages.

Goats are of significant economic importance, yet our knowledge of the molecular pathways involved in their pregnancy remains limited. This study aims to investigate the role of IGFs in uterine and ovarian cellular events during pregnancy in goats. Forty-two Hair Goats were examined, including four pregnancy groups representing embryo-positive (G1, n=7), early (G2, n=7), mid (G3, n=7), and late pregnancy (G4, n=7), as well as two luteal stage groups representing early (G5, n=7) and late (G6, n=7) phases. Uterine and ovarian tissues were collected, and RT-qPCR and immunohistochemistry were performed to evaluate IGF expression. The results showed that IGF1 and IGF2 expressions were significantly higher in G1 than in other pregnancy and control groups (p < 0.05). Additionally, IGFBP1 expression was higher in G2 than in G1 and G4 (p < 0.05), and IGFBP3 expression was higher in G4 than in any other pregnancy stage (p < 0.05). However, no statistically significant differences were observed in the expression levels of IGFBP4 and IGFBP6 between any of the groups. Finally, IGFBP5 expression was significantly higher in G1, G3, and G4 compared to G2 (p < 0.05). Overall, the dynamic changes observed in the expression of the IGF gene family during different stages of pregnancy highlight the crucial role of IGFs in regulating pregnancy in goats.

Differential expression of angiogenesis-related genes in goat uterus and corpus luteum during pregnancy.

Vascularization and the control of luteal and endometrial development are regulated by hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) during pregnancy. In this study, the mRNA and protein expression levels of HIFs (HIF1A, HIF2A and HIF3A) and VEGF in goat uterine and ovarian tissues during various stages of pregnancy were evaluated. A total of 42 Hair goats were used and were allocated into six groups, namely embryo-positive (G1), early pregnancy (G2), mid-term pregnancy (G3), late pregnancy (G4), oocyte-positive group (G5) and diestrus group (G6). The mRNA expression of the examined genes was evaluated by RT-qPCR, and protein expression was evaluated by immunohistochemistry (IHC). In caruncles, HIF1A mRNA expression was greater in G1, G2 and G4 than in G3 (p < .05). HIF1A and HIF2A expression was greater in G1 than in G5 (p < .05). In cotyledons, HIF1A, HIF2A and HIF3A mRNA expression was greater in G2 and G3 compared to G4 (p < .05). In luteal tissue, HIF1A mRNA expression was greater in G1 and G2 than in G3 and G4 (p < .05). In the immunohistochemical examination, HIF1A, HIF2A, HIF3A and VEGF immunoreactions were detected in uterine and luteal tissues. Findings suggest that HIFs and VEGF are involved in the regulation of ovarian functions as well as the processes of implantation and placentation.

The efficacy of EMG-biofeedback training on quadriceps muscle strength in patients after arthroscopic meniscectomy.

AIMS: In 40 patients, we attempted to investigate the efficacy of electromyography-biofeedback (EMG-B) on quadriceps muscle strength after arthroscopic meniscectomy. METHODS: The patients were randomly divided into two groups each consisting of 20 subjects. For the control group, a classical exercise program was given (five sessions of EMG-B application for 2 weeks postoperatively). Range of motions, Lysholm knee score, EMG electrical activity values of vastus medialis obliques (VMO), and vastus lateralis (VL) were measured pre- and postoperatively on the 3rd and 14th day, and at the 6th week. RESULTS: When the ranges of motion values were compared, a significant difference (for average values of knee flexion angle) was found on the 14th day and 6th week in favour of biofeedback group (p<0.05). When Lysholm knee scores on the 14th day and 6th week were compared in the control and biofeedback groups, and maximum contraction and average contraction values of VMO, VL muscles were compared with operated/non-operated %age ratios, there was a statistically significant difference in favour of the biofeedback group (p<0.05). CONCLUSIONS: Our results showed that EMG-B was an effective treatment modality in improving quadriceps muscle strength after arthroscopic meniscectomy surgery.