The association of thyroid autoimmunity with ovarian reserve in women with type 1 diabetes with and without polycystic ovary syndrome.

The aim of the study was to investigate the relation between thyroid autoimmunity (TAI), reflected as the presence of thyroid peroxidase antibodies (TPOAb), and parameters of ovarian reserve in women with type 1 diabetes (T1DM) and polycystic ovary syndrome (PCOS). We studied 83 euthyroid women with T1DM (age - 26 ± 5 years, BMI - 24 ± 3 kg/m2) - 12 with PCOS and positive TPOAb (PCOS + TPOAb), 29 with PCOS with negative TPOAb (PCOS + noTPOAb), 18 without PCOS with positive TPOAb (noPCOS + TPOAb), 24 without PCOS with negative TPOAb (noPCOS + noTPOAb). Serum concentrations of anti-Müllerian hormone (AMH), sex hormones, TSH, thyroid hormones and TPOAb were assessed. The prevalence of TAI was comparable between PCOS and noPCOS. We did not observe differences in hormonal profile or AMH concentration between two PCOS groups-PCOS + TPOAb and PCOS + noTPOAb (p > 0.05). Women with PCOS + TPOAb had lower FSH concentration and higher LH/FSH index than noPCOS + noTPOAb (p = 0.027; p = 0.019, respectively). Moreover, PCOS + TPOAb had lower oestradiol level than noPCOS + TPOAb (p = 0.041). AMH concentration was higher in both groups with PCOS, independent of TPOAb presence, than in noPCOS + noTPOAb (both p < 0.001). The presence of positive TPOAb titre was not related to the studied parameters of ovarian reserve - AMH and ovarian follicle number. In multiple linear regression analysis, the only significant predictor of AMH in the whole studied group with T1DM was total daily insulin dose U/kg (ß = - 0.264; p = 0.022). The presence of TAI did not affect the hormonal profile or ovarian reserve in women with T1DM with and without PCOS.

Polymorphic Variants of Genes Encoding Angiogenesis-Related Factors in Infertile Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a global health issue affecting a significant number of infertile women who undergo in vitro fertilization (IVF) cycles. Extensive vasculogenesis and angiogenesis occur in both maternal and fetal placental tissues, and vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family molecules and their receptors are potent angiogenic mediators in the placenta. Five single nucleotide polymorphisms (SNPs) in the genes encoding angiogenesis-related factors were selected and genotyped in 247 women who had undergone the ART procedure and 120 healthy controls. Genotyping was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A variant of the kinase insertion domain receptor (KDR) gene (rs2071559) was associated with an increased risk of infertility after adjusting for age and BMI (OR = 0.64; 95% CI: 0.45-0.91, p = 0.013 in a log-additive model). Vascular endothelial growth factor A (VEGFA) rs699947 was associated with an increased risk of recurrent implantation failures under a dominant (OR = 2.34; 95% CI: 1.11-4.94, padj. = 0.022) and a log-additive model (OR = 0.65; 95% CI 0.43-0.99, padj. = 0.038). Variants of the KDR gene (rs1870377, rs2071559) in the whole group were in linkage equilibrium (D' = 0.25, r2 = 0.025). Gene-gene interaction analysis showed the strongest interactions between the KDR gene SNPs rs2071559-rs1870377 (p = 0.004) and KDR rs1870377-VEGFA rs699947 (p = 0.030). Our study revealed that the KDR gene rs2071559 variant may be associated with infertility and rs699947 VEGFA with an increased risk of recurrent implantation failures in infertile ART treated Polish women.

Ibuprofen Reduces Testosterone Level in Women With Polycystic Ovary Syndrome.

Context: Hyperandrogenism is a central feature of polycystic ovary syndrome (PCOS). In vitro studies have demonstrated that inflammatory stimuli promote whereas ibuprofen inhibits androgen production by ovarian theca-interstitial cells. Objective: This work aimed to determine the effects of nonselective inhibitor of cyclooxygenases COX-1 and COX-2 on testosterone levels. Methods: A prospective pilot study took place in an academic hospital of women with PCOS defined according to Rotterdam criteria (N = 20). Evaluations were taken at baseline and after 3 weeks of ibuprofen administration (400 mg twice a day or 400 mg 3 times a day, respectively, in women with weight < and ≥ 70 kg). The main outcome measure was total serum testosterone. Results: Ibuprofen administration was associated with a decline of total testosterone from 0.75 ±â€…0.06 ng/mL to 0.59 ±â€…0.05 ng/mL (P = .008). There was no statistically significant change in the levels of other relevant hormones including dehydroepiandrosterone sulfate, gonadotropins, and insulin. Multiple regression analysis identified the greatest decline of testosterone was independently predicted by baseline testosterone level (P = .004) and by baseline insulin sensitivity index (P = .03). Conclusion: Nonselective inhibition of COX-1 and COX-2 leads to selective reduction of testosterone consistent with direct inhibitory effect on ovarian steroidogenesis.

Fetomaternal Expression of Glucose Transporters (GLUTs)-Biochemical, Cellular and Clinical Aspects.

Several types of specialized glucose transporters (GLUTs) provide constant glucose transport from the maternal circulation to the developing fetus through the placental barrier from the early stages of pregnancy. GLUT1 is a prominent protein isoform that regulates placental glucose transfer via glucose-facilitated diffusion. The GLUT1 membrane protein density and permeability of the syncytial basal membrane (BM) are the main factors limiting the rate of glucose diffusion in the fetomaternal compartment in physiological conditions. Besides GLUT1, the GLUT3 and GLUT4 isoforms are widely expressed across the human placenta. Numerous medical conditions and molecules, such as hormones, adipokines, and xenobiotics, alter the GLUT's mRNA and protein expression. Diabetes upregulates the BM GLUT's density and promotes fetomaternal glucose transport, leading to excessive fetal growth. However, most studies have found no between-group differences in GLUTs' placental expression in macrosomic and normal control pregnancies. The fetomaternal GLUTs expression may also be influenced by several other conditions, such as chronic hypoxia, preeclampsia, and intrahepatic cholestasis of pregnancy.

[Corrigendum] Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Following the publication of this paper, the authors have requested that, on p. 4412 of the above article in the Funding section of the Declarations, the acknowledgement to one of the funding sources should be removed from the paper; essentially, the reference to grant no. 2018/31/B/NZ5/02475, formulated by the Polish National Science Centre (grant providing institution), should be removed from the paper. Therefore, the revised version of the Funding section paragraph should read as follows: Funding: The present study was supported by a grant from Poznan University of Medical Sciences (grant no. 502­14­02227367­10694). The authors confirm that there are no further errors in the study, and all the authors agree to this correction. The authors are grateful to the Editor of Molecular Medicine Reports for granting them this opportunity to publish a Corrigendum, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 20: 4403-4414, 2019, DOI: 10.3892/mmr.2019.10709].

Serum Metabolomics in PCOS Women with Different Body Mass Index.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine and metabolic disorder, affecting 5-10% of women of reproductive age. It results from complex environmental factors, genetic predisposition, hyperinsulinemia, hormonal imbalance, neuroendocrine abnormalities, chronic inflammation, and autoimmune disorders. PCOS impacts menstrual regularities, fertility, and dermatological complications, and may induce metabolic disturbances, diabetes, and coronary heart disease. Comprehensive metabolic profiling of patients with PCOS may be a big step in understanding and treating the disease. The study aimed to search for potential differences in metabolites concentrations among women with PCOS according to different body mass index (BMI) in comparison to healthy controls. We used broad-spectrum targeted metabolomics to evaluate metabolites' serum concentrations in PCOS patients and compared them with healthy controls. The measurements were performed using high-performance liquid chromatography coupled with the triple quadrupole tandem mass spectrometry technique, which has highly selective multiple reaction monitoring modes. The main differences were found in glycerophospholipid concentrations, with no specific tendency to up-or down-regulation. Insulin resistance and elevated body weight influence acylcarnitine C2 levels more than PCOS itself. Sphingomyelin (SM) C18:1 should be more intensively observed and examined in future studies and maybe serve as one of the PCOS biomarkers. No significant correlations were observed between anthropometric and hormonal parameters and metabolome results.

The Influence of Prepubertal Onset of Type 1 Diabetes and Age of Menarche on Polycystic Ovary Syndrome Diagnosis.

CONTEXT: Higher prevalence of polycystic ovary syndrome (PCOS) in women with type 1 diabetes (T1DM) is linked to exogenous insulin, especially when diabetes is diagnosed before puberty. OBJECTIVE: The study evaluates the impact of prepubertal onset of T1DM and insulin therapy on PCOS diagnosis and phenotypic characteristics in women with T1DM. DESIGN, SETTING, AND PATIENTS: We studied 83 women with T1DM (age 26 ± 5 years, BMI 24 ± 3 kg/m2) 36 with premenarchal (PM) onset of T1DM [17 with PCOS diagnosed (PCOS+PM) and 19 without PCOS (noPCOS+PM)] and 47 women with postmenarchal onset of T1DM [24 with PCOS (PCOS-noPM) and 23 without PCOS (noPCOS-noPM)]. OUTCOME MEASUREMENTS: Clinical examination, assessment of serum sex hormones, glycated hemoglobin (HbA1c) and ultrasonographic evaluation of the ovaries were performed in all women. RESULTS: Applying Rotterdam criteria, 49% of women with T1DM were diagnosed with PCOS. There were no differences in hormonal profile and ovarian parameters between PCOS+PM and PCOS-noPM. Women with T1DM+PM had higher insulin dose/24 h and U/kg bw/24 h than T1DM-noPM (P-values = 0.014 and 0.001, respectively). Both PCOS+PM and noPCOS+PM groups had higher insulin dose U/kg bw/24 h in comparison to PCOS-noPM (P-values = 0.004 and = 0.006, respectively). In multivariable logistic regression analysis, age of menarche [odds ratio (OR): 0.672; 95% confidence interval (CI): 0.465-0.971] and HbA1c (OR: 0.569; 95% CI: 0.383-0.846) were associated with the diagnosis of PCOS. CONCLUSIONS: There were no differences in the prevalence of PCOS between T1DM+PM and T1DM-noPM; however, earlier menarche might have an influence on PCOS diagnosis in women with T1DM.

Heterogeneity of Endocrinologic and Metabolic Parameters in Reproductive Age Polycystic Ovary Syndrome (PCOS) Women Concerning the Severity of Hyperandrogenemia-A New Insight on Syndrome Pathogenesis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, anovulation, infertility, obesity, and insulin resistance, which results in increased concentrations of testosterone (T), which disturbs follicular growth and ovulation. This study aimed to assess PCOS women's clinical, endocrinological, and metabolic parameters concerning hyperandrogenism severity. RESULTS: 314 women (mean age 27.3 ± 4.6; mean body mass index (BMI) 25.7 ± 5.6) with PCOS, were divided into terciles according to T concentrations: <0.64 ng/mL (group 1), 0.64 to 0.84 ng/mL (Group 2) and >0.84 ng/mL (group 3). The mean concentration of T in all women was 0.59 ng/mL and correlated negatively with the number of menstrual cycles per year (MPY) (r = -0.36; p < 0.0001) and positively with Ferriman-Gallway score (FG) (r = 0.33; p < 0.0001), luteinizing hormone (LH) (r = 0.19; p < 0.0001) and dehydroepiandrosterone sulfate (DHEAS) (r = 0.52; p < 0.0001). Positive correlation between BMI and hirsutism (r = 0.16; p < 0.0001), total cholesterol (TC) (r = 0.18; p < 0.0001), low-density lipoprotein (LDL) (r = 0.29; p < 0.0001), and triglycerides (TG) (r = 0.40; p < 0.0001) was demonstrated. The division into subgroups confirmed the lowest MPY, highest LH, and hirsutism in group 3. BMI, insulin sensitivity indices, and lipid profile parameters were not different between the three T subgroups. CONCLUSIONS: We found no correlation between testosterone levels and insulin sensitivity or dyslipidemia in women with PCOS. Metabolic abnormalities may contribute more significantly than hyperandrogenemia to PCOS development.

Human Wharton's Jelly-Cellular Specificity, Stemness Potency, Animal Models, and Current Application in Human Clinical Trials.

Stem cell therapies offer a great promise for regenerative and reconstructive medicine, due to their self-renewal and differentiation capacity. Although embryonic stem cells are pluripotent, their utilization involves embryo destruction and is ethically controversial. Therefore, adult tissues that have emerged as an alternative source of stem cells and perinatal tissues, such as the umbilical cord, appear to be particularly attractive. Wharton's jelly, a gelatinous connective tissue contained in the umbilical cord, is abundant in mesenchymal stem cells (MSCs) that express CD105, CD73, CD90, Oct-4, Sox-2, and Nanog among others, and have the ability to differentiate into osteogenic, adipogenic, chondrogenic, and other lineages. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs) do not express MHC-II and exhibit immunomodulatory properties, which makes them a good alternative for allogeneic and xenogeneic transplantations in cellular therapies. Therefore, umbilical cord, especially Wharton's jelly, is a promising source of mesenchymal stem cells.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well­known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT­qPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNA­dependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

Relationship between adipocytokines and angiotensin converting enzyme gene insertion/deletion polymorphism in lean women with and without polycystic ovary syndrome.

This study was designed to investigate the relationship between the levels of select adipocytokines (adiponectin, visfatin and apelin) and angiotensin in converting enzyme (ACE) gene insertion/deletion (ID) polymorphism in lean women with and without polycystic ovary syndrome (PCOS). The PCOS group (N = 94) was identified according to the Rotterdam criteria. The Control group (N = 68) included age- and body mass index (BMI)-matched healthy volunteers. Serum levels of adipocytokines were measured using enzyme immunoassays (EIA) and ACE genes were evaluated by polymerase chain reaction (PCR). The PCOS group, when compared to the Control group had lower adiponectin (p < .001) but higher visfatin (p < .001) and apelin (p = .003). There was no significant correlation of the levels of these adipocytokines with BMI, fasting glucose, fasting insulin or Homeostasis Model Assessment-Insulin Resistance (HOMA-IR). The PCOS and the Control groups also differed with regard to the ACE ID genotype distribution (p < .001). The ID, DD, and II genotype frequencies were, respectively, 34, 57 and 9% in the PCOS group and 49, 22 and 29% in the Control group. When stratified according to individual ID genotypes, the levels of adipocytokines in the PCOS and the Control groups remained significantly different. There was no statistically significant relationship between the levels of adipocytokines and ACE ID genotypes.

Novel markers of human ovarian granulosa cell differentiation toward osteoblast lineage: A microarray approach.

Under physiological conditions, human ovarian granulosa cells (GCs), are responsible for a number of processes associated with folliculogenesis and oogenesis. The primary functions of GCs in the individual phases of follicle growth are: Hormone production in response to follicle stimulating hormone (FSH), induction of ovarian follicle atresia through specific molecular markers and production of nexus cellular connections for communication with the oocyte. In recent years, interest in obtaining stem cells from particular tissues, including the ovary, has increased. Special attention has been paid to the novel properties of GCs during long­term in vitro culture. It has been demonstrated that the usually recycled material in the form of follicular fluid can be a source of cells with stem­like properties. The study group consisted of patients enrolled in the in vitro fertilization procedure. Total RNA was isolated from GCs at 4 time points (after 1, 7, 15 and 30 days of culture) and was used for microarray expression analysis (Affymetrix® Human HgU 219 Array). The expression of 22,480 transcripts was examined. The selection of significantly altered genes was based on a P­value <0.05 and expression higher than two­fold. The leucine rich repeat containing 17, collagen type I α1 chain, bone morphogenetic protein 4, twist family bHLH transcription factor 1, insulin like growth factor binding protein 5, GLI family zinc finger 2 and collagen triple helix repeat containing genes exhibited the highest changes in expression. Reverse­transcription­quantitative PCR was performed to validate the results obtained in the analysis of expression microarrays. The direction of expression changes was validated in the majority of cases. The presented results indicated that GCs have the potential of cells that can differentiate towards osteoblasts in long­term in vitro culture conditions. Increased expression of genes associated with the osteogenesis process suggests a potential for uninduced change of GC properties towards the osteoblast phenotype. The present study, therefore, suggests that GCs may become an excellent starting material in obtaining stable osteoblast cultures. GCs differentiated towards osteoblasts may be used in regenerative and reconstructive medicine in the future.

Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence-A Transcriptomic Study.

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

Genes Involved in the Processes of Cell Proliferation, Migration, Adhesion, and Tissue Development as New Potential Markers of Porcine Granulosa Cellular Processes In Vitro: A Microarray Approach.

Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.

Is copeptin a new potential biomarker of insulin resistance in polycystic ovary syndrome?

O: BJECTIVES: Copeptin has been reported to play an important role in metabolic response in women with PCOS. However, the optimal cut-off value for detecting subjects with insulin resistance (IR) remains undetermined. We investigated whether copeptin can serve as an indicator of IR and tried to determine the optimal cut-off value of plasma copeptin concentration in detecting subjects with PCOS and IR. MATERIAL AND METHODS: We carried out a case-control study on 158 women with PCOS and HOMA-IR < 2.5, 96 women with PCOS with HOMA-IR ≥ 2.5, and 70 healthy volunteers. Plasma copeptin, as well as hormonal, biochemical, metabolic, and IR parameters, were measured. To investigate whether copeptin allows IR to be predicted in PCOS, we used logistic regression models and ROC curve analysis. RESULTS: Median plasma copeptin concentration was the highest in the women with PCOS and HOMA-IR ≥ 2.5. Logistic regression analysis revealed that copeptin was the strongest predictor of HOMA ≥ 2.5 (OR: 53.34 CI 7.94-358.23, p < 0.01). Analysis of ROC curves indicated that the cut-off value above 4 pmol/L of plasma copeptin concentration had high (99%) specificity but very low (21%) sensitivity in diagnosing of IR (AUC 0.607 (95% CI 0.53-0.68. CONCLUSIONS: Our findings suggest that copeptin is associated with IR in PCOS patients, but due to low sensitivity should not be considered as a marker of IR.

Continuous subcutaneous insulin infusion reduces neonatal risk in pregnant women with type 1 diabetes mellitus.

OBJECTIVES: An attempt was made to demonstrate the superiority of the treatment model using continuous subcutaneous insulin infusion (CSII) over multiple daily injections (MDI) of insulin in achieving a successful pregnancy outcome and good newborn's condition in patients with type 1 diabetes. MATERIAL AND METHODS: The study included 297 infants born to type 1 diabetic patients; 175 patients were treated with MDI and 122 with CSII. Maternal metabolic control during pregnancy, gestational weight gain, insulin requirements, pregnancy outcome and neonatal status were compared between MDI and CSII arm. The composite adverse neonatal outcome was diagnosed if at least one of the following was found: abnormal birth weight (LGA or SGA), congenital malformation, miscarriage, intrauterine fetal death, emergency CS due to fetal risk, iatrogenic prematurity, RDS, hypoglycemia, hyperbilirubinemia, and the postpartum pH in the umbilical artery ≤ 7.1. RESULTS: The studied groups did not differ regarding gestational week at delivery, a proportion of births at full term, preterm births, miscarriages, or late pregnancy losses (intrauterine fetal death > 22 weeks). Newborns of mothers treated with CSII showed lower incidence of neonatal complications (composite adverse neonatal outcome) compared to those of mothers treated with MDI (60% vs 74%, respectively; p = 0.01). We did not find any association between the mode of treatment and composite adverse maternal outcome. CONCLUSIONS: The use of CSII in the treatment of pregnant women with type 1 diabetes was associated with reduced number of neonatal complications presented as neonatal composite outcome but had no influence on maternal outcome.

New Gene Markers of Angiogenesis and Blood Vessels Development in Porcine Ovarian Granulosa Cells during Short-Term Primary Culture In Vitro.

The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of "angiogenesis," "blood vessel development," "blood vessel morphogenesis," "cardiovascular system development," and "vasculature development" for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC's in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.

'Heart development and morphogenesis' is a novel pathway for human ovarian granulosa cell differentiation during long­term in vitro cultivation­a microarray approach.

Granulosa cells (GCs) have many functions in the endocrine system. Most notably, they produce progesterone following ovulation. However, it has recently been proven that GCs can change their properties when subjected to long­term culture. In the present study, GCs were collected from hyper­stimulated ovarian follicles during in vitro fertilization procedures. They were grown in vitro, in a long­term manner. RNA was collected following 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to validate the obtained results. Two ontological groups characteristic for processes associated with the development and morphogenesis of the heart were identified during the analyses: 'Heart development' and 'heart morphogenesis'. The results of the microarrays revealed that the highest change in expression was demonstrated by the lysyl Oxidase, oxytocin receptor, nexilin F­actin binding protein, and cysteine­rich protein 3 genes. The lowest change was exhibited by odd­skipped related transcription factor 1, plakophilin 2, transcription growth factor­ß receptor 1, and kinesin family member 3A. The direction of changes was confirmed by RT­qPCR results. In the present study, it was suggested that GCs may have the potential to differentiate towards other cell types under long­term in vitro culture conditions. Thus, genes belonging to the presented ontological groups can be considered as novel markers of proliferation and differentiation of GCs towards the heart muscle cells.

Genes responsible for proliferation, differentiation, and junction adhesion are significantly up-regulated in human ovarian granulosa cells during a long-term primary in vitro culture.

The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups "cell differentiation" (GO:0030154), "cell proliferation" (GO:0008283) and "cell-cell junction organization" (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs' in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.