Abstract: The objective was to evaluate the effects of different culture systems and the addition of fibroblast growth factor (bFGF) during in vitro culture (IVC) of testicular tissue fragments from prepubertal collared peccaries. Testes from five individuals were collected, dissected, and cultured for up to 56 days (34°C and 5% CO2) in Dulbecco's Modified Eagle's Medium (DMEM), supplemented or not with FGF at 10 ng/mL, in organotypic (ORG) or 3D system culture. Samples were evaluated every 14 days for histomorphology, cell viability, DNA integrity, and proliferative activity. Overall, the ORG system without FGF addition was the best to preserve testicular fragment histomorphology, viability, and DNA integrity during IVC. However, the 3D system, regardless of the presence of FGF, impaired the DNA integrity of testicular cells in all culture periods analyzed. Regarding cell proliferation, at 14 days the ORG group without addition of FGF showed a percentage of Ki-67 positive cells indicative of proliferation similar to the non-cultured group, while the other treatments reduced proliferation. However, at 28 days a reduction in proliferation was observed in this same group and an increase in proliferation in the others. Cell proliferation was reduced in all groups at 42 days (P < 0.05). In summary, we suggest the use of the organotypic system for long-term culture of testicular fragments of prepubertal collared peccaries. In addition, FGF supplementation to the culture medium does not seem to be essential.
Lay summary: Animals do not produce sperm cells before puberty. However, in case of unexpected death of young individuals carrying critical genes for the diversity and sustainability of an animal population, sperm cells can be obtained by recovering and culturing tissue from the testes in proper laboratory conditions. Resulting sperm cells can then be used to produce embryos using IVF methods. The goal of the present work was to find the best culture conditions to keep pieces of testicular tissue alive for extended periods of time using the collared peccary as a model. Two different methods were tested. The first approach was to place a piece of tissue on top of a gel that was rich in nutrients, similar to the natural supply to the tissue. This method is called organotypic culture. The second approach was to recreate a more natural environment by embedding the tissue inside the gel, which is known as 3D culture. Overall, the organotypic culture was the best way to keep the tissues alive for 56 days. This is a major step forward to allow the production of sperm cells from peccaries in the laboratory.
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Abstract: Over the past 30 years, the number of new cases of uterine leiomyomas (UL) in women of reproductive age has increased by 67.14% worldwide. The limitations of the current therapeutic options have led to the search for alternatives. Myrianthus arboreus P. Beauv., used for infertility and tumors, has never been tested for UL. In the present study, the decoction of its stem bark was evaluated in a model of UL induced in female Wistar rats. Animals were treated once daily by gavage for 30 days. Normal control and model groups received distilled water, positive groups received mifepristone (5.2 mg/kg) and the remaining three groups were treated with 50, 100, and 200 mg/kg M. arboreus extract, respectively. Compared to the model group, the extract at 50 and 100 mg/kg reduced the E2B- and progesterone-induced uterine horn asymmetry and thickening, and the relative uterine weight and diameter; myometrial thickness; and collagen density (at 100 and 200 mg/kg). Regarding cytokines, the extract decreased the uterine levels of TGF-β1 and VEGF (at 100 and 200 mg/kg) and TNF-α (at all doses tested). It also decreased the serum levels of estradiol (at 100 and 200 mg/kg). Despite positive trend in reducing oxidative damage (decreased MDA at 50 mg/kg, increased catalase activity at all tested doses, and increased GSH at 100 mg/kg), the level of oxidative stress is still elevated. By attenuating key cellular events involved in the growth and development of UL, such as inflammation, fibrosis and angiogenesis, Myrianthus arboreus may be a promising option for UL treatment and management.
Lay summary: Uterine fibroids are non-cancerous tumors that originate from the smooth muscular wall of the uterus. They may cause severe symptoms, such as chronic pelvic pain, heavy menstrual bleeding, painful intercourse, frequent urination, back pain and obstetric complications (embryo implantation failure, fetal growth restriction, miscarriages, preterm delivery, and fertility impairment). The exact cause is not known, but hormonal changes may play an important role. Treatment options depend on the symptoms and desire to preserve the uterus, and may include medication, minimally invasive procedures and surgery. Their associated risks and limitations fuel searching for conservative alternatives. This study investigates the anti-uterine fibroid potential of the giant yellow mulberry (Myrianthus arboreus P. Beauv.) in rat. Consuming boiled M. arboreus stem bark in water reduced uterine wall thickness, inflammation, and collagen deposition, and increased the ability of the uterus to fight cell damage. Thus, this extract may prevent or shrink uterine fibroids by reducing uterine swelling, fibrosis and excessive cell growth.
Abstract: Ghrelin, which is a hormone composed of 28 amino acids that is mainly produced in the stomach, is also secreted by Leydig cells in the testes of rats and humans. The hypothalamus regulates testosterone secretion by releasing gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gland to release luteinizing hormone (LH). LH then prompts the testes to produce testosterone via the activity of steroidogenic acute regulatory protein (StAR). Consequently, ghrelin may play a regulatory role in gonadal function. Male Sprague-Dawley rats were randomly assigned to four groups: the control, ghrelin-treated, diabetic, and diabetic plus ghrelin treatment groups. After the rats were sacrificed, plasma samples were collected. Leydig cells were isolated and cultured with human chorionic gonadotropin (hCG, which is similar to LH and is used to stimulate Leydig cells to synthesize testosterone), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP, which is an activator of cyclic adenosine monophosphate-dependent protein kinase), or forskolin (an activator of adenylyl cyclase in a wide variety of cell types). Compared with normal treatment, ghrelin treatment in diabetic rats markedly increased plasma testosterone levels by 3.75-fold (P < 0.05), Leydig cell testosterone secretion by 2.8-fold (P < 0.05), GnRH-mediated LH release from the anterior pituitary by 2.95-fold (P < 0.05), and StAR expression by 1.96-fold (P < 0.05) in testicular Leydig cells. These findings indicated that ghrelin enhanced testosterone production in diabetic rats, which was partially achieved by the hypothalamic-pituitary-gonadal axis and StAR. This study emphasized the potential use of ghrelin as a treatment for improving testosterone levels and gonadal function in individuals with diabetes.
Lay summary: This study explored how a hormone known as ghrelin (which is mainly produced in the stomach) may help in regulating testosterone levels. Researchers examined male rats in four groups, including diabetic rats treated with ghrelin, and discovered that ghrelin increased testosterone production in diabetic rats by improving the communication between the brain, pituitary gland, and testes. This hormone also helped specific cells in the testes to function more effectively. The diabetic rats treated with ghrelin exhibited notable increases in testosterone levels and improved hormone function. These findings suggest that ghrelin could potentially help to address hormonal imbalances related to diabetes and improve reproductive health. This research highlights the potential benefits of ghrelin in addressing hormonal imbalances.
Abstract: Debate persists regarding the optimal endometrial preparation model for frozen embryo transfer (FET). Among the various approaches, the natural cycle and artificially programmed cycles are commonly employed. However, no established guidelines currently recommend a preferred method. The aim of the present study was to compare live birth rates after FET in natural cycle versus artificial cycle endometrial preparation in a non-selected, real-life population. This retrospective study included patients from a single centre who underwent vitrified-thawed blastocyst transfer between January 2016 and April 2023. In the natural cycle FET group, no medication was used, and the transfer date was determined by luteinising hormone ovulation test results. In the artificial cycle FET group, patients received oestradiol and progesterone tablets. A total of 905 cycles were analysed, which included 164 NC-FET cycles and 741 AC-FET cycles. From the 295 live births, there were a total of 320 neonates, with multiple gestations occurring in 8% of cases. The live birth rates were significantly higher in the NC-FET group at 43%, compared to 30% in the AC-FET group (P = 0.001). The AC-FET group also experienced higher rates of biochemical pregnancies and spontaneous abortions. However, when adjusting for confounding variables in multivariate analysis, the type of FET was not found to be an independent predictor of live birth.
Conclusion: Our findings suggest that while NC-FET is associated with higher live birth rates, other factors such as patient characteristics also play a significant role in these differences. Further prospective studies are needed to validate these results.
Abstract: In South Africa, approximately 10% of the calculated need for medically assisted reproduction is being met due to limited access and unequal availability of these services. To facilitate understanding of challenges associated with access to assisted reproduction, a retrospective case study spanning 6 years was performed at one public sector hospital in South Africa offering these services. Demographic profiles, including income, region of residency and access to medical insurance, of patients seeking assistance to become pregnant were investigated. Patients were categorised as those who underwent diagnostic investigations only vs those who returned for therapeutic procedures, and the difference in demographic profiles between the two groups was determined. This investigation showed that patients from the lower-income classification group, without medical insurance, tend to return for therapeutic procedures less often than those with a higher income and medical insurance, even though these low-income patients qualify for a therapeutic procedure subsidy. An inverse relationship existed where patient numbers decreased as their travel distance increased, but patients who were required to travel further for assisted reproductive therapy tended to return for these procedures more often than patients who resided closer to the medical facility. In conclusion, access to medically assisted reproduction facilities is critically undersupplied and limited in the region. In order to ease the travel distance of patients, alternative primary diagnostic routes with accessible clinics are needed. In addition, costs of therapeutic procedures in the public sector should be re-evaluated to be offered at affordable rates for marginalised patients.
Lay summary: In South Africa, about 10% of patients who need assistance to become pregnant are being helped. To better understand this phenomenon, researchers considered information about patients from a public sector hospital in South Africa. This includes how much money the patients earned, how far they travelled to the hospital and whether they had medical insurance. The patients were grouped into those who requested initial investigations but never returned for treatments, and those who returned for medical treatment. The differences between these groups were then evaluated. The research showed that people with less money tend to abandon further treatment more often, or take longer to return, than those with more money. The conclusion drawn is that assisted reproductive therapy is too expensive and that more IVF clinics are needed, using cheaper and simpler procedures of the same quality.
Abstract: Development and function of the uterus and placenta are essential for a successful pregnancy, particularly in livestock species where reproductive efficiency is economically important. Several metabolic pathways play critical roles in uterine and placental function during pregnancy by supporting processes important for cellular function and proliferation, as well as conceptus growth and development. Among these, one-carbon metabolism, the pentose cycle, serine biosynthesis (serinogenesis), and polyamine metabolism have emerged as key metabolic pathways contributing to uterine and placental function that enhance conceptus growth. These pathways are not only regulated by maternal plane of nutrition but also by stage of the estrous cycle or day of gestation, implying that circulating steroid hormones may influence metabolism through these pathways. This review first discusses the development of the female reproductive system and the placenta, focusing on sheep, cattle, and pigs. We then highlight what is currently known regarding key metabolic pathways in the uterus and placenta of these species and where knowledge gaps still exist. Improving our understanding of the mechanisms that regulate the metabolism of key nutrients provides a basis for nutritional or hormonal interventions that can potentially improve pregnancy success and conceptus development in livestock species.
Lay summary: Key nutrients are essential for a successful pregnancy. These nutrients are processed through several important pathways that support the growth and function of the uterus and placenta. Understanding the impacts of these pathways may help improve pregnancy outcomes in farm animals. This review summarizes our current knowledge on the impact of maternal nutrition and hormonal status on key pathways for the development and function of the uterus and placenta.
Abstract: Despite recent improvements in equine ovum pick-up (OPU) combined with intracytoplasmic sperm injection (ICSI), there is still significant inter-individual variability. In this study, serum concentrations of the oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), as well as the GDF9/BMP15 complex, anti-Müllerian hormone (AMH), progesterone (P4), and free and conjugated estradiol-17β (E2), were determined in 59 mares (3-24 years) at the time of OPU. Mares were classified retrospectively, based on the number of embryos obtained, into low or high outcome groups, using two or three embryos as the threshold values. Results of OPU-ICSI were not affected by the mare's age, body condition score, cycle stage, number of aspirated follicles, number of recovered oocytes, or the recovery rate (P > 0.05 in all cases). Mares with <2 or ≥2 embryos had similar hormone levels (P > 0.05 in all cases), but there was a >90% likelihood of obtaining ≥2 embryos per session when P4 and E2 concentrations were considered simultaneously. Mares with ≥3 embryos/session had lower P4 and higher AMH (P < 0.05 in both cases). GDF9 and BMP15 were detectable in 14 and 12% of mares, respectively, while the GDF9/BMP15 complex was detected in 93% of mares, with no correlation to other factors. In conclusion, these preliminary findings suggest a negative correlation between high P4 levels and OPU-ICSI outcome in horses.
Lay summary: We analyzed the relationship between the hormone profiles of mares at the time of egg collection and the outcome of intracytoplasmic sperm injection (ICSI), as ICSI success often varies significantly between individuals. There were no differences in the hormone profiles of mares producing fewer than two or two or more embryos. However, mares with three or more embryos/session showed significant differences in their hormone profiles compared to those producing fewer than three embryos/session. These changes were mainly related to steroid hormones produced by the ovary, such as progesterone and estradiol, and to the concentration of AMH. Our results suggest a possible influence of the hormone profile of the mare on the number of embryos obtained.
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Abstract: The sperm freezing-thawing procedure is the most commonly used technique in clinics to preserve male fertility before any pathological destruction of the testis. Therefore, most studies are currently focused on optimizing this method to achieve high-quality semen after thawing. During cryopreservation, oxidative stress-induced damage affects sperm structures and decreases their fertility potential. The use of antioxidants in freezing media can protect sperm against oxidative damage. We designed this study to evaluate whether incubation of semen with human follicular fluid, which contains a wide variety of enzymatic and nonenzymatic antioxidants, can prevent the negative effects of freezing-thawing on human spermatozoa. Human semen was divided into three groups i) the 0-hour group (before freezing), ii) the control group (after freezing-thawing), and iii) the FF group (after freezing with 50% follicular fluid). The sperm motility, viability, integrity of the plasma membrane and DNA, mitochondrial membrane potential, malondialdehyde level, total antioxidant capacity, and catalase activity were assessed in these three groups. The findings showed a significant decrease in sperm motility, viability, plasma membrane and DNA integrity, mitochondrial membrane potential, total antioxidant capacity, and catalase activity and a significant increase in malondialdehyde level in the control group compared with the 0-hour group. The FF group displayed a considerable increase in sperm parameters, total antioxidant capacity, and catalase activity and a significant decrease in malondialdehyde level compared with the control group. Follicular fluid can be considered an effective supplement to improve antioxidant indices and sperm parameters during freezing-thawing.
Lay summary: Sperm freezing is a useful method in clinics to preserve fertility in people who are affected by some problems such as diseases or chemotherapy which decrease their fertility. Although various studies are focused on optimizing this method, some challenges decrease the efficiency of this method. Oxidative stress has been reported as one of the mechanisms inducing negative effects on sperm during freezing-thawing. Therefore, the use of cryoprotectants and also some antioxidants has been suggested to increase sperm quality during freezing-thawing. In this study, we used human follicular fluid before freezing to assess sperm parameters. Our results showed that follicular fluid with antioxidant properties and other proper factors can have positive effects on human sperm during freezing-thawing and could be proposed to be added to the sperm freezing medium to improve sperm parameters, although this suggestion needs to be confirmed by further experiments.
Abstract: Tissue non-specific alkaline phosphatase (TNSALP) regulates postnatal phosphate homeostasis, but its role in utero-placental phosphate availability remains poorly understood. Gilts were bred and hysterectomized on Day 60 or Day 90 of gestation (n = 6/day). Phosphate was less abundant in allantoic and amniotic fluids on Day 90 compared to Day 60. TNSALP protein was immunolocalized, and enzymatic activity was quantified and localized in endometrial and chorioallantois tissues. Day had no effect on TNSALP activity in the chorioallantois. In contrast, endometrial TNSALP activity was lower on Day 90 compared to Day 60. Phosphate abundance in allantoic fluid correlated positively with endometrial TNSALP activity on Day 60 but not Day 90. TNSALP protein was abundantly expressed in the endometrium and chorioallantois on both days investigated, with localization to the endometrial, chorionic, and areolar epithelia, as well as stromal cells and endothelium. TNSALP activity was detected in the endothelium of the blood vessels in both the endometrium and chorioallantois, and on the basal surface of the endometrial glands on Day 60 but not Day 90. The endometrial stratum compactum stroma had strong TNSALP activity on Day 60. Weak TNSALP activity was present in the areolar epithelium, with a modest increase in activity on Day 90 compared to Day 60. TNSALP activity was present in the columnar chorionic epithelial cells, with an apparent decrease in activity in the chorioallantois on Day 90 compared to Day 60. These data reveal spatiotemporal changes in TNSALP localization and activity, suggesting its involvement in regulating phosphate availability at the utero-placental interface in swine.
Lay summary: Phosphate is an essential nutrient for fetal growth, but how it is managed during pregnancy is not fully understood. This study explored the role of an enzyme called tissue non-specific alkaline phosphatase (TNSALP) in regulating phosphate availability in the uterus and placenta in pigs in mid- and late pregnancy. Phosphate levels decreased in the fluids surrounding the fetus in late pregnancy. TNSALP was present in the uterus and placenta, and the amount of the enzyme varied depending on the tissue and stage of pregnancy and correlated with changes in phosphate levels. These findings suggest that TNSALP plays a key role in managing phosphate transport from the mother to the fetus in pregnancy to support fetal development.
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