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Abstract

Genetic diversity and the optimal genetic composition are essential for the adaptability and adaptation of tree populations. Artificial regeneration of stands might reduce the genetic diversity and increase family structures if the seeds were collected from a limited number of mother trees. We did a genetic inventory in 12 pedunculate oak stands in Russia using a set of 366 nuclear gene markers (361 SNPs, 5 Indels) in order to look for differences in the genetic composition among natural and artificial stands. Our results did not reveal any systematic differences among both types of stands. However, we found two extreme cases of limited genetic diversity and increased proportion of full-sibs and half-sibs in urban man-made stands. The implications for the forestry and gene conservation programs were discussed.

Abstract

The human granulosa cells (GCs) surround the oocyte and form the ovarian follicle’s proper architecture.

These sub-populations include mural granulosa cells, antral granulosa cells, and cumulus granulosa cells.

Their main functions are to support the oocyte’s growth (cumulus granulosa cells) and estradiol production (mural granulosa cells). After ovulation, the granulosa cells transform into the luteal cells of the corpus luteum and produce progesterone.

Our study investigated the expression profile of three genes: TGFB1, CD105, and FSP1 during a 7-day in vitro culture. The analysis was conducted using the RT-qPCR technique.

Changes in the expression of CD105 and FSP1 could be observed during the 7-day in vitro culture. In the case of TGFB, the expression remained at a similar level, with no statistically significant differences observed.

Running title: Expression of TGFB1, CD105 and FSP1 in granulosa cells

Abstract

Ovarian granulosa cells (GCs) play a crucial role in oocyte maturation, creating a favorable microenvironment around the oocyte. Therefore, enhanced apoptosis and GCs loss may negatively affect the intra-follicular milieu and compromise the oocyte quality, reducing pregnancy chances. Based on the RT-qPCR method, the present research revealed the differential expression of apoptosis-related genes (BCL2, BAX, p53, CASP9) during the seven days of primary in vitro culture of GCs isolated from patients undergoing in vitro fertilization (IVF) procedure. Individual gene expression changes may reflect the GCs survival and/or apoptotic status at different time points.

Running title: Apoptosis-related genes expression in granulosa cells in vitro

Abstract

Sperm separation for ICSI is an essential step in realization of the IVF procedures. The method of microfluidic separation of sperm cells using chips has been applied more and more frequently in recent years. This method is often presented as extremely gentle to spermatozoa and decreasing significantly concentration of sperm cells with fragmented DNA when compared to conventional methods. The aim of our study was to verify a microfluidic chip system from the perspective of its potential to select spermatozoa with non-fragmented DNA. We tested the efficiency of this separation method against the swim-up method. In this study we evaluated sperm DNA integrity before and after the separation methods in ten patients. Ejaculate of each patient was separated by both the swim up method and the microfluidic chip method at the same time. It was shown that both the methods are very similar in reduction of spermatozoa with fragmented DNA. Interestingly, the concentration of spermatozoa with fragmented DNA was lower after the microfluidic separation than after the swim-up method in all the patients. Nevertheless, the differences were not statistically significant with only 2.1% on average, which is negligible in terms of practical use.

Running title: Microfluidic chip and DNA fragmentation

Abstract

Gestational diabetes mellitus (GDM) is thought to be the most common metabolic gestational complication. Its prevalence has been continuously increasing in recent decades along with the rising epidemic of obesity in modern societies. GDM is associated with an increased risk of fetal growth abnormalities, birth traumas, and several neonatal complications. Widely available screening tools, fasting glucose measurements, combined with oral glucose tolerance test results, contribute to the reduction of the risk of those complications. Nevertheless, we are still looking for novel reliable early markers of GDM. It has been established that high 1st-trimester exosome concentrations could predispose the development of GDM in later pregnancy. Exosomes can be easily isolated from various tissues and body fluids in pregnant patients. Due to this, extracellular vesicle concentration assessment appears as a new promising tool in the prediction of GDM at the preclinical stage of the disease. Furthermore, it has been found that women already diagnosed with GDM have significantly higher exosome concentrations compared with healthy individuals. These findings could help to elucidate the molecular pathogenesis of GDM. Exosomes are loaded with various molecules especially proteins, lipids, mRNAs, and microRNAs. Altered expression of numerous microRNAs and enzymes such as dipeptidyl peptidase-IV in exosomes isolated from patients with GDM may suggest their direct contribution to the mechanisms of glucose intolerance. This knowledge could be used in the development of new therapeutic strategies in patients with GDM. Nevertheless, it should be emphasized that these are only preliminary results that require further investigations.

Running title: Exosomes in gestational diabetes

Abstract

The essential function of granulosa cells is to maintain the proper course of oogenesis and folliculogenesis.

The immune system is an additional local regulator of ovarian function, with cytokines necessary for the proper function of the ovaries, including the secretion of steroid hormones

This study aimed to analyze the expression of genes in human GCs in short-term primary culture and define the difference in the expression of IL1β, IL6, and TNFα genes at 48h and 72h of culture compared to the 24h control. Total RNA was isolated using the Chomczyński and Sacchi protocol. RNA samples were treated with DNase I and reverse transcribed (RT) into cDNA. The determination of transcript levels of the mentioned genes was performed using the Light Cycler® 96 Real-Time PCR kit, Roche Diagnostics GmbH (Mannheim, Germany).

The present study proved that granulosa cells in a short-term primary in vitro culture express IL-1β, IL-6, and TNFα. The tested genes show a decrease in expression at 24h of culture and a subsequent slight increase at 72h, not exceeding the initial levels. The expression changes the most for IL1β and the least for TNFα.

The fluctuations in the amount of transcript may be influenced by factors stored in granulosa cells before the IVM procedure, the procedure of in vitro fertilization, as well as factors related to the process of primary culture. More research is needed to understand the details of these occurrences.

Running title: The inflammatory response in human granulosa cells

Abstract

The human placenta is a complex, multifunctional transient fetomaternal organ. The placenta is composed of the maternal decidua basalis and its fetal part, consisting of the mesenchymal and trophoblast cell lineages. Both the placenta and the amniotic membranes are abundant in readily available placenta-derived mesenchymal stem cells (PD-MSCs). The clinical application of the PD-MSCs opens new perspectives for regenerative medicine and the treatment of various degenerative disorders. Their properties depend on their paracrine activity – the secretion of the anti-inflammatory cytokines and specific exosomes. In contrast to the PD-MSCs, the trophoblast stem cells (TSCs) are much more elusive. They can only be isolated from the blastocyst-stage embryos or the first-trimester placental tissue, making that procedure quite demanding. Also, other cultures require specific, strictly controlled conditions. TSCs may be potentially used as an in vitro model of various placental pathologies, facilitating the elucidation of their mysterious pathogenesis and creating the environment for testing the new drug efficiency. Nonetheless, it is unlikely that they could be ever implemented as a part of novel cellular therapeutic strategies in humans.

Running title: Current knowledge on the placental stem cells

Abstract

ADSCs are readily accessible and widely available. Isolated through a minimally invasive procedure from adipose depots, they can be found at diverse body location, where they served various functions, including energy homeostasis. They can be obtained upon surgeries from otherwise waste tissues, like after excision of fat tissue or liposuction. In addition, due to the possibility to isolate many ADSCs, in vitro proliferation can be performed in a short time period, resulting in cells showing more predictable results[]. For this study ADSCs were obtained from waste material following routing sterilization procedures of dogs. This study aimed to analyse the expression of MSC specific markers before and after in vitro differentiation of ASCs. Three positive and three negative markers were analysed, CD105, CD73, CD90, CD34, CD14 and CD45. There were significant differences detected in the expression of all of the genes, with most of them exhibiting notable downregulation. The only exception, CD14 showed major upregulation after the process of differentiation. These changes confirm the success of differentiation, as well as suggest that this process significantly lowers the stem-like ability of ASCs. This knowledge should serve as a reference for further molecular and clinical studies, possibly aiding the understanding of the internal mechanisms governing the differentiation and stemness of ASCs, to enable their widespread and safe application in regenerative medicine.

Running title: Mesenchymal markers during ASC osteogenic differentiation

Abstract

Due to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.

Running title: ASC marker expression during long-term in vitro culture