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Chromosomal abnormalities represent a significant genetic cause of male infertility because they impair spermatogenesis. The objective of the current study was to determine the prevalence and distribution of chromosomal abnormalities in Greek men with infertility. Four hundred eighty-eight infertile men (27 azoospermic, 168 with oligospermia -98 mild, 57 moderate, 13 severe- and 293 with normospermia) undergoing In Vitro Fertilization (IVF) between 2016-2022 were enrolled in the study. Thirty-eight fertile men were also studied. Chromosomal analysis of peripheral blood lymphocytes was performed using standard cytogenetic techniques. 21/488 (4.3%) of men tested had an abnormal karyotype; 13 (2.7%) had sex chromosome abnormalities and 8 (1.6%) had autosomal ones. No chromosomal aberration was detected in the control group. The frequency of chromosomal alterations was significantly higher in azoospermic men than in men with oligospermia and normospermia (37% vs 4.2% and 1.4% respectively, p < 0.05). Moreover, in men with oligospermia, cytogenetic abnormalities were more common in the severe group (7.7%) followed by the moderate (5.25%) and the mild group (3%). The results of the study are by the literature. Karyotyping is suggestive especially in oligospermic /azoospermic men and before proceeding to IVF. The advent of high throughput sequencing technologies and genome-wide association studies will contribute to discovering novel promising genetic factors involved in male infertility.

Background: In view of the current interest in exploring the clinical use of mesenchymal stem cells (MSCs) from different sources, we performed a side-by-side comparison of the biological properties of MSCs isolated from the Wharton's jelly (WJ), the most abundant MSC source in umbilical cord, with bone marrow (BM)-MSCs, the most extensively studied MSC population.Methods: MSCs were isolated and expanded from BM aspirates of hematologically healthy donors (n = 18) and from the WJ of full-term neonates (n = 18). We evaluated, in parallel experiments, the MSC immunophenotypic, survival and senescence characteristics as well as their proliferative potential and cell cycle distribution. We also assessed the expression of genes associated with the WNT- and cell cycle-signaling pathway and we performed karyotypic analysis through passages to evaluate the MSC genomic stability. The hematopoiesis-supporting capacity of MSCs from both sources was investigated by evaluating the clonogenic cells in the non-adherent fraction of MSC co-cultures with BM or umbilical cord blood-derived CD34+ cells and by measuring the hematopoietic cytokines levels in MSC culture supernatants. Finally, we evaluated the ability of MSCs to differentiate into adipocytes and osteocytes and the effect of the WNT-associated molecules WISP-1 and sFRP4 on the differentiation potential of WJ-MSCs.Results: Both ex vivo-expanded MSC populations showed similar morphologic, immunophenotypic, survival and senescence characteristics and acquired genomic alterations at low frequency during passages. WJ-MSCs exhibited higher proliferative potential, possibly due to upregulation of genes that stimulate cell proliferation along with downregulation of genes related to cell cycle inhibition. WJ-MSCs displayed inferior lineage priming and differentiation capacity toward osteocytes and adipocytes, compared to BM-MSCs. This finding was associated with differential expression of molecules related to WNT signaling, including WISP1 and sFRP4, the respective role of which in the differentiation potential of WJ-MSCs was specifically investigated. Interestingly, treatment of WJ-MSCs with recombinant human WISP1 or sFRP4 resulted in induction of osteogenesis and adipogenesis, respectively. WJ-MSCs exhibited inferior hematopoiesis-supporting potential probably due to reduced production of stromal cell-Derived Factor-1α, compared to BM-MSCs.Conclusions: Overall, these data are anticipated to contribute to the better characterization of WJ-MSCs and BM-MSCs for potential clinical applications.

We have previously shown (Batsali A et al., Blood 2013:122, 1212) that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. Provided that the WNT-pathways are involved in proliferation and differentiation of BM-MSCs, we assumed that the aforementioned findings might be attributed, at least in part, to aberrant WNT-signaling in WJ-MSCs. In support of this hypothesis, we found that gene expression of the Wnt antagonist sFRP4, a promoter of adipogenesis in human adipose tissue-derived MSCs, was significantly down-regulated in WJ-MSCs and that mRNA levels of WNT-induced secreted protein-1, (WISP-1), a regulator of osteogenesis in BM-MSCs, were also significantly reduced in WJ-MSCs. These observations imply a connection between these WNT-associated molecules and the biological properties of WJ-MSCs, which requires, however, further investigation. The present study was undertaken so as to explore the effects of WISP-1 and sFRP4 in growth and differentiation of ex-vivo expanded WJ-MSCs. MSCs were isolated from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=5) after written informed consent of the family. MSCs were in vitro expanded, re-seeded for a total of 4 passages (P) and phenotypically characterized by flow cytometry at P3. WJ-MSCs were grown in the absence or presence of rhWISP-1 or rhsFRP4 and cell proliferation was assessed by a methyl-triazolyl-tetrazolium (MTT)-assay. In addition, WJ-MSCs were induced to differentiate in vitro to osteoblasts and adipocytes, in the absence or presence of rhWISP-1 or rhsFRP4 respectively. Differentiation was quantified by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes by real time RT-PCR. Relative gene expression was calculated by the ΔCt method. The expression of WISP-1 and sFRP4 by non-differentiated WJ- and BM-MSCs as well as by WJ-MSCs during osteogenesis and adipogenesis, respectively, was also evaluated by real time RT-PCR. Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. In line with our previous work WISP-1 and sFRP4 mRNA were significantly decreased in WJ-MSCs, compared to BM-MSCs. To explore the role of WISP-1 in WJ-MSCs' growth we cultured cells in the presence of 50 ng/ml or 100 ng/ml rhWISP1 and assessed cell proliferation at multiple time points, throughout a 14-day culture. WISP-1 treatment did not lead to any significant effect in cell numbers. Next, we investigated the time course of WISP1 gene expression during osteoinduction. In all samples, WISP1 mRNA levels increased during osteogenesis. As compared to day0 (exposure to osteogenic medium), the increase in gene expression reached statistical significance at days 7 and 14. Furthermore, WISP-1 gene expression was significantly higher at day 14, compared to day 7. To investigate the functional effects of WISP1 on the osteoblastic differentiation of WJ-MSCs, cells were cultured for 7 days in osteogenic medium supplemented with 50ng/ml rh-WISP1. A significant increase in the expression of RUNX2 and ALP was detected, compared to non-treated cells. To investigate the impact of sFRP4 in WJ-MSC's proliferation we exposed cells to 20nM rhsFRP4 for 14 days. Live cell numbers, at various time points, were significantly reduced in treated cells. Regarding the time course of sFRP4 expression during adipogenic differentiation, sFRP4 mRNA levels increased during adipogenesis reaching statistical significance at days7 and 14, as compared to day0. In addition, sFRP4 gene expression was significantly higher at day 14 as compared to day 7. Finally, when cells underwent adipogenic differentiation in the presence of rhSFRP4, a significant increase in PPARG and CEBPA mRNA levels was detected at day 14, as compared to non-treated cells Collectively, our results suggest that WISP-1 and sFRP4 may be actively implicated in proliferation and differentiation of WJ-MSCs. The functional role of these WNT-related molecules in the biology of WJ-MSCs requires deeper understanding, in view of the growing interest for the use of WJ-MSCs in cell-based therapies. Disclosures No relevant conflicts of interest to declare.

Bone narrow (BM)- derived mesenchymal stem/stromal cells (MSCs) represent the most extensively studied population of adult MSCs and are considered as the gold-standard for MSC-based clinical applications. Yet, it is now becoming increasingly clear that BM may not represent the most suitable source for MSC collection. Indeed, Umbilical cord (UC) has emerged as a more abundant and easily attainable source of MSCs and several reports have shown that MSCs can be efficiently isolated from the connective tissue that surrounds UC vessels, namely the Wharton's jelly (WJ). According to the existing literature, WJ-MSCs display typical MSC characteristics, however a head-to-head comparison with BM-MSCs is still lacking. Provided that ex vivo MSC expansion is a prerequisite for clinical MSC-applications, in the present study we seek to comparatively investigate the characteristics of WJ- and BM-MSCs, cultured under identical conditions. MSCs were isolated and expanded from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=10) after written informed consent of the family. MSCs were in vitro expanded and re-seeded for a total of 10 passages (P) and phenotypically characterized by flow cytometry (FC). MSCs were induced to differentiate in vitro to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. Relative gene expression was calculated by the ΔCt method. MSC growth characteristics were assessed by evaluating the population doubling time (DT) and by a methyl-triazolyl-tetrazolium (MTT)-assay throughout passages. Cell-cycle analysis was performed using propidium iodide (PI) staining. MSC survival was evaluated by FC with 7-Aminoactinomycin D (7-AAD) and senescence was estimated by the percentage of SA-b-gal+ cells in cultures. Moreover, MSC karyotypic stability was assessed with classic G-banding. Finally the expression of genes related to Wnt-mediated signal transduction was also investigated, using a PCR array. Total RNA was thus isolated from 6 representative BM- and 6 WJ-MSC cultures at P2. The fold change (FC) for each gene between the group of WJ- and the group of BM-MSCs was calculated with the ΔΔCt method (FC=2-ΔΔCt). WJ-MSCs displayed a spindle-shape morphology, similar to BM-MSCs. Furthermore, WJ- and BM-MSCs displayed identical immunophenotype, as evidenced by the expression of CD90,CD105,CD44,CD29,CD73 and the lack of expression of CD45,CD14,CD34,CD31. WJ-MSCs displayed superior proliferative potential compared to BM-MSCs throughout passages (p Taken together WJ-MSCs display decreased cellular senescence after extended in vitro culture, increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, as compared to BM-MSCs. The last two observations can be explained, at least partly, by the aberrant expression of Wnt-signaling molecules in WJ-MSCs. The emerging role of Wnt-signaling pathway in WJ-MSC biology is currently under investigation. Disclosures: No relevant conflicts of interest to declare.