Radioactive Iodine-induced hypothyroidism interferes with the maturation of reproductive organs during puberty in immature female rats.

Animal and human studies suggest that thyroid hormone may have critical roles in the development of the ovary. For example, thyroid deficiency disrupts the ovarian microarchitecture and menstrual cycle in neonate and adult women, respectively. Therefore, it is conceivable that thyroid deficiency might disrupt sexual maturation during the peri-pubertal period. To investigate the impact of radioactive iodine-induced thyroid deficiency on reproductive organs throughout puberty, immature female rats were given water containing radioactive iodine (0.37 MBq/g body weight) twice, on postnatal days 22 and 29. Radioactive iodine-induced hypothyroidism was revealed by low free thyroxin levels. Thyroid deficiency delayed the onset of vaginal opening, reduced ovarian weight and the number of medium-sized follicles and led to elongated uteri. However, there was no effect on the estrous cycle or absolute uterus weight. We conclude that radioactive iodine-induced thyroid deficiency delays sexual maturation and alters normal ovarian growth in peri-pubertal rats.

Excretory-Secretory Products of Trichomonas vaginalis Cause Apoptosis in Mouse Sperm in Vitro.

Excretory-secretory products (ESP) of T. vaginalis have been shown to inhibit sperm motility, viability, and functional integrity, leading to a decreased fertilization rate in vitro. This study investigated whether T. vaginalis induce apoptosis and ultrastructural changes of sperm using flow cytometry and electron microscopy. Incubation of sperm with T. vaginalis ESP increased phosphatidylserine externalization and DNA fragmentation, and decreased mitochondrial membrane potential. Transmission electron microscopy of sperm incubated with ESP revealed abnormal features such as distorted heads, broken necks, and acrosomes exocytosis. This is the first report that demonstrates a direct impact of T. vaginalis ESP on sperm apoptosis and architecture in vitro.

Red cell manufacturing using parallel stirred-tank bioreactors at the final stages of differentiation enhances reticulocyte maturation.

The aim of this study was to develop a robust, quality controlled, and reproducible erythroid culture system to obtain high numbers of mature erythroblasts and red blood cells (RBCs). This was achieved using a fully controlled stirred-tank bioreactor by the design of experiments (DOE) methods in the serum-free medium by defining the appropriate culture parameters. Human cord blood CD34+ cells were first cultured in static flasks and then inoculated to stirred-tank bioreactors. Cell diameter was gradually decreased and final RBC yields were significantly higher when cells were inoculated at sizes smaller than 12 µm. The larger immature cells in the basophilic stage did not survive, while smaller mature erythroid cells were successfully expanded at high agitation speeds, demonstrating that appropriate seeding timing is critical. A high inoculation cell density of 5 × 106 cells/ml was achieved reaching 1.5 × 107 cells/ml. By using DOE analysis fitted to precise stages of erythropoiesis, we were able to acquire the optimal culture parameters for pH (7.5), temperature (37°C), dissolved oxygen, agitation speed (500 rpm), inoculation timing (cell diameter 12-13 µm), media feeding regimen, and cell seeding density (5 × 106 cells/ml). The final pure RBCs showed appropriate functions compared with fresh donor RBCs, confirming that manufacturing mature RBCs with reproducibility is possible.

The Effects of High Peripubertal Caffeine Exposure on the Adrenal Gland in Immature Male and Female Rats.

The consumption of high levels of dietary caffeine has increased in children and adolescents. Human and animal studies have shown that chronic intake of high doses of caffeine affects serum glucocorticoid levels. Given that glucocorticoids play a role in peripubertal organ growth and development, chronic high doses of caffeine during puberty might impair maturation of the adrenal glands. To evaluate any effects of caffeine exposure on growing adrenal glands, 22-day-old male (n = 30) and female Sprague Dawley rats (n = 30) were divided into three groups (n = 10/group); group 1 received tap water (control) and groups 2 and 3 received water containing 120 and 180 mg/kg/day caffeine, respectively, via gavage for 4 weeks. At the end of the experiment, adrenal glands were weighed and processed for histological analysis. Relative adrenal weights increased in both groups of caffeine-fed males and females, whereas absolute weights were decreased in the females. In the female caffeine-fed groups the adrenal cortical areas resembled irregularly arranged cords and the medullary area was significantly increased, whereas no such effects were seen in the male rats. Our results indicate that the harmful effects of caffeine on the adrenal glands of immature rats differ between females and males. Although female rats seemed to be more susceptible to damage based on the changes in the microarchitecture of the adrenal glands, caffeine affected corticosterone production in both female and male rats. In addition, increased basal adrenocorticotropic hormone levels in caffeine-fed groups may reflect decreased cortical function. Therefore, caffeine may induce an endocrine imbalance that disturbs the establishment of the hypothalamo-pituitary adrenal axis during puberty, thereby leading to abnormal stress responses.

Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by downregulating Cyp19A1 expression.

The transition from granulosa cell (GC) to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 (Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone (LH)/human chorionic gonadotropin treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore, we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory GCs obtained from pregnant mare serum gonadotropin-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG-Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and chromatin immunoprecipitation assays. The results revealed that the steroidogenic factor-1 (SF1)-binding motif, but not the specificity protein 1 (Sp1) binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs and that it has an essential role in the luteal transition in steroidogenesis.

Improvement of Red Blood Cell Maturation In Vitro by Serum-Free Medium Optimization.

Despite recent studies on media additives, the low viability and dysplastic features of terminally mature erythroid cells are still major problems in enhancing erythroid cell yields in vitro. Moreover, research on enhancing terminal erythropoiesis has been focused on the immature stage of erythroid cells such as burst forming unit-erythroid and colony forming unit-erythroid. Here, we tested many commercially available serum-free culture media and developed a superior culture media formulation compared with the conventional control for higher expansion-fold, higher viability, and therefore enhanced red cell productivity. The addition of the specific medium to the previously known best media at a specific ratio, whose effects were not dose-dependent, enabled the generation of significantly higher erythrocyte products with over 1.3 million-fold proliferation of erythroid cells after maintenance for 21 days throughout the maturation stages from CD34+ cells. The cells cultured in this condition expressed maturation markers and were significantly superior in differentiation and enucleation. Comparative mRNA profiling revealed that erythroid cells in this medium showed more efficient maturation in mRNA levels. The cultured cells showed comparable erythroblast survival and also restored better red blood cell (RBC) functions of oxygen saturation profile with expression of adult globin up to 99%. However, to develop chemically defined media, the well-known supplements including hormones, cytokines, and serum-replacement reagents were not sufficient to replace the optimized media in producing mature RBCs. Taken together, our optimized medium formulation under serum-free culture conditions produced the best reproducible results on productivity and maturation in erythroid cells with economic benefits. These culture conditions may thus serve as a useful platform for further investigation of in vitro erythropoiesis and to develop defined serum-free media for clinical trials.

Nonintegrating Direct Conversion Using mRNA into Hepatocyte-Like Cells.

Recently, several researchers have reported that direct reprogramming techniques can be used to differentiate fibroblasts into hepatocyte-like cells without a pluripotent intermediate step. However, the use of viral vectors for conversion continues to pose important challenges in terms of genome integration. Herein, we propose a new method of direct conversion without genome integration with potential clinical applications. To generate hepatocyte-like cells, mRNA coding for the hepatic transcription factors Foxa3 and HNF4α was transfected into mouse embryonic fibroblasts. After 10-12 days, the fibroblasts converted to an epithelial morphology and generated colonies of hepatocyte-like cells (R-iHeps). The generated R-iHeps expressed hepatocyte-specific marker genes and proteins, including albumin, alpha-fetoprotein, HNF4α, CK18, and CYP1A2. To evaluate hepatic function, indocyanine green uptake, periodic acid-Schiff staining, and albumin secretion were assessed. Furthermore, mCherry-positive R-iHeps were engrafted in the liver of Alb-TRECK/SCID mice, and we confirmed FAH enzyme expression in Fah1RTyrc/RJ models. In conclusion, our data suggest that the nonintegrating method using mRNA has potential for cell therapy.

Effect of radioactive iodine-induced hypothyroidism on longitudinal bone growth during puberty in immature female rats.

Thyroid cancer in children, the most common endocrine malignancy, shows aggressive behavior and has a high recurrence rate after surgical ablation. Radioactive iodine (RAI) treatment is the most effective primary modality for medical ablation of juvenile thyroid cancer, and leads to intentional hypothyroidism. Although several negative impacts of hypothyroidism have been reported in children in response to other antithyroid agents, the combined effects of RAI exposure and hypothyroidism, on growing bones specifically, are unknown. In this study, we investigated the effect of RAI-induced hypothyroidism on the long bones during the pubertal growth spurt using immature female rats. Female Sprague-Dawley rats were randomly divided into a control group, and an RAI-treated group fed with RAI (0.37 MBq/g body weight) twice via gavage. After 4 weeks, we observed a significantly-reduced serum free thyroxine level in the RAI group. The latter group also displayed decreased body weight gain compared to the control. In addition, the lengths of long bones, such as the leg bones and vertebral column, as well as bone mineral content, were reduced in the RAI-treated animals. Our results confirm the negative impacts of RAI-induced thyroid deficiency during puberty on longitudinal bone growth and bone mineralization.

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

Comparison of fetal heart rate patterns using nonlinear dynamics in breech versus cephalic presentation at term.

BACKGROUND: It has been reported that breech fetuses have inferior neurological outcomes regardless of mode of delivery, raising the possibility that in utero neurological impairment is more frequent in breech fetuses, possibly contributing to malpresentation. AIMS: To assess differences between the cardiovascular autonomic nervous systems (ANSs) of breech and cephalic fetuses using nonlinear dynamic indices of fetal heart rate (FHR) variability. STUDY DESIGN AND SUBJECTS: This study included 86 fetuses with breech presentation and 173 fetuses with cephalic presentation, with no other maternal or fetal problems. We analyzed FHR variability and spectral indices as markers of ANS behavior. We used nonlinear dynamic indices to represent the complexity of heart rate regulation, as well as correlation dimension as a chaotic index of the cardiovascular control system. RESULTS: One of FHR parameters (Mean minute range) was significantly lower in breech than cephalic fetuses (p=0.0294). However, there were no other significant differences in any linear or nonlinear indices, nor in clinical outcomes, between breech and cephalic fetuses. CONCLUSION: Our data suggest that breech fetuses have neither more active ANS nor less active complexity control systems than do cephalic fetuses. This indicates that the neurologic maturation of breech fetuses is not inferior to cephalic ones. The practical implication of these findings is that the nervous system integrity of breech fetuses may not result directly in neonatal complications.

Expression of endothelial nitric oxide synthase in the uterus of patients with leiomyoma or adenomyosis.

AIM: To confirm the difference in the expression of endothelial nitric oxide synthase in the normal endometrium and myometrium of women who have leiomyoma or adenomyosis compared with controls, and its correlation with the pathogenesis of menorrhagia or dysmenorrhea in patients with uterine leiomyoma. METHODS: Fifty-one hysterectomized patients were divided into three groups: (i) patients with leiomyoma (n=24); (ii) those with adenomyosis (n = 19); and (iii) the control group (n=8). The expression of endothelial nitric oxide synthase was confirmed on immunohistochemistry and analyzed using an evaluation nomogram. RESULTS: The expression of endothelial nitric oxide synthase was significantly higher in the leiomyoma group and the adenomyosis group as compared with the control group. In the subgroup analysis of leiomyoma depending on symptoms (menorrhagia or dysmenorrhea or both), the expression of endothelial nitric oxide synthase was significantly higher in the symptomatic subgroup than the asymptomatic subgroup (endometrium P=0.0029, myometrium P=0.0276). CONCLUSIONS: Based on the findings that the expression of endothelial nitric oxide synthase was significantly higher in the uterus with leiomyoma or adenomyosis, it can therefore be inferred that nitric oxide might have a pathological effect on the uterus with the above diseases. In particular, it is also presumed that endothelial nitric oxide synthase is closely associated with menorrhagia and dysmenorrhea.

Spontaneous uterine rupture in the first trimester: a case report.

Uterine rupture is one of the most feared obstetric complications affecting the pregnant woman and fetus. Most of the cases have various risk factors and mainly occur during the second or third trimester. However, spontaneous uterine rupture during the first trimester is extremely rare. We experienced a case of spontaneous uterine rupture in a 36-yr-old multiparous woman without definite risk factors. The initial impression was a hemoperitoneum of an unknown origin with normal early pregnancy. Intensive surgical method would be needed for accurate diagnosis and immediate management in bad situation by hemoperitoneum even though a patient was early pregnancy.