In vitro models aiming for fertility preservation

Fertility preservation is considered a great hope to people suffering from infertility as a consequence of developmental or genetic cause, a disease itself or its treatments sequelae. In vitro models are fundamental to enrich our knowledge about human germ cell development and its regulation, to generate and screen new therapeutic measures and might provide a tool that can be transferred to clinical use to treat infertility. There are, in principle, two strategies to start an in vitro fertility preservation protocol in male humans. First the in vitro generation of germ cells by differentiating human pluripotent stem cells and second the maturation and differentiation of human spermatogonia stem cells into more advanced stage of germ cells. However, both strategies are not suitable to be used in the clinic since the robust protocols for differentiation are not available. Additionally, reproducibility of the results between different laboratories is a big challenge faced by researchers working in the field. Therefore, we focused in this thesis on these two strategies aiming to improve both models to reduce variability and improve reproducibility. In the first study, we addressed the variability occurring among hES cell lines when used for human germ cell differentiation. We aimed to find a suitable culture condition allowing a robust starting point for our cultures by using LN521 as a culture matrix for hES cells before using them in protocols of differentiation. Moreover, we investigated the only robust working system in rodents and aimed to translate this to the situation in humans by using normal fetal gonadal tissues as well as actual patient materials to investigate the potential use of these systems to mature human germ cells in vitro. We found that LN521 has positive effects on hES cells growth and maintenance of their pluripotency characteristics with no influence on gonadal cells related genes expression. Furthermore, we showed that LN521 homogenized the gene expression variation among the five cell lines used in the study. In regard to in vitro germ cells maturation from spermatogonial stem cells, production of elongated spermatids was achieved when air liquid interphase organ culture method was used to cultivate mouse testicular tissues in vitro. A supplementation of 10% knockout serum replacement is shown to have positive effects on tubular maturation, germ cell proliferation and differentiation. We showed that organ culture method could be used to culture and study human first trimester gonads somatic cells in vitro as demonstrated by their ability to produce hormones in a manner similar to what is described for in vivo situation. In addition, differences in somatic cells functions in testicular tissues from different patient groups exposed to hematological and oncological treatments could be illustrated by culturing these tissues in vitro by using organ culture method. Culturing human embryonic stem cells on LN521 could be a step forward towards future applications of human embryonic stem cells in regenerative medicine by providing more predictable and controllable system to assess the behavior of human embryonic stem cells when used in different protocols for differentiation. This would be the basis for future approaches to generate more defined in vitro protocols for differentiation of human pluripotent stem cells towards germ cells. On the other hand, organ culture method could be a useful tool to study the process of human germ cells development and to screen the effect of different substances on human gonadal cell development and function. Moreover, it can be used as quality assessment tool of cryopreserved testicular tissues, from patients exposed to hematological and oncological treatments, before considering using them for fertility preservation purposes

Anatomical variations of the frontal sinus: A computed tomography-based study [version 2; peer review: 2 approved]

Background: The pneumatization of the frontal sinus is variable between individuals, including monozygotic twins. The volumetric anatomic variants of the frontal sinus are classified into aplasia, hypoplasia, medium-sized, and hyperplasia. We aimed to study the frontal sinus morphology in Omani patients using computed tomography (CT) evaluations. Methods: Retrospectively, 1220 frontal sinus CT scans from 610 patients investigated at Sultan Qaboos University Hospital, Oman, from January 2019 to December 2020 were reviewed. The frontal sinus morphology was classified according to the classification proposed by Guerram et al. The Chi-square test was used to determine the influence of sex. Results: With regard to the unilateral occurrence, the most prevalent frontal sinus category observed was medium-sized (13.3%), followed by hyperplasia (7.9%), hypoplasia (5.4%), and aplasia (2%) categories. Similarly, in bilateral occurrence, the most common frontal sinus category observed was medium-sized (53%), followed by hyperplasia (13.1%), hypoplasia (3.4%) and aplasia (2%) categories. Right and left frontal sinus aplasia were observed in 2.1% and 1.8% of cases, respectively. In terms of sex influence, the left unilateral (p<0.01) and the bilateral hypoplasia (p<0.05) were significantly higher in females. On the other hand, the left unilateral (p<0.01) and the bilateral hyperplasia (p<0.05) were higher in males. Conclusions: The baseline data of frontal sinus category frequencies reported in the present study is helpful in the diagnostic evaluation of sinusitis in the clinical setting. The preoperative recognition of frontal sinus types, particularly frontal sinus aplasia in multiplanar CT scans, is crucial to avoid unexpected complications while performing endoscopic sinus surgery

Laminin 521 Stabilizes the Pluripotency Expression Pattern of Human Embryonic Stem Cells Initially Derived on Feeder Cells

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells

Human Pluripotent Stem Cells in Reproductive Science—A Comparison of Protocols Used to Generate and Define Male Germ Cells from Pluripotent Stem Cells

Globally, fertility-related issues affect around 15% of couples. In 20%&ndash;30% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used

Students&rsquo; Performance in Face-to-Face, Online, and Hybrid Methods of Teaching and Assessment in Anatomy

In recent times, online teaching and assessment have provided a great opportunity to explore better methods in medical education. There are inconsistent views concerning the effectiveness of online assessment. Hence, the present study aimed to evaluate online teaching and assessment methods in relation to face-to-face methods by comparing students&rsquo; performances. The students&rsquo; assessment results in two basic anatomy courses, which are part of the Doctor of Medicine and Biomedical Sciences programs at Sultan Qaboos University, were analysed. We compared the students&rsquo; mean scores and coefficient of variance in the multiple-choice written exams and the objective structured practical exams during the spring semesters of 2019, 2020, and 2021, containing face-to-face teaching and exams, partial online teaching and online exams, and online teaching and both proctored online and face-to-face exams, respectively. The sudden transition to online teaching and assessment halfway through the semester resulted in higher means and a lower coefficient of variance among students&rsquo; scores in both theory and practical exams. However, when the fully adopted online method of teaching and assessment was employed, the mean scores decreased, and the coefficient of variance increased to figures close to those witnessed before the pandemic, when teaching and assessment were face-to-face. This trend applied to both the Doctor of Medicine and Biomedical Sciences programs&rsquo; anatomy courses. The results indicate that online assessment of theoretical and practical anatomical knowledge is comparable to that of face-to-face assessment. However, proper planning and preparedness are mandatory to achieve the desired outcomes

Laminin 521 Stabilizes the Pluripotency Expression Pattern of Human Embryonic Stem Cells Initially Derived on Feeder Cells

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells

Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells

<div><p>The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells <i>in vitro</i> and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, <i>in vitro</i> germ cell differentiation, and <i>in vivo</i> germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of <i>OCT4</i>, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both <i>in vitro</i> and <i>in vivo</i>.</p></div

Xenotransplantation of transfected cells into the seminiferous tubules of mouse testes.

<p>A) Weight of the xenografts collected 8 weeks after transplantation, n ≥ 3. B) Whole mount staining with an antibody against primate testis cells. A cell clump was observed in one <i>pbNANOS3</i> transplanted testis. Six colonies with spermatogonial characteristics were observed in one <i>pbDAZL</i> transplanted testis, representative image shown. Scale bar: 100 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s009" target="_blank">S4 Table</a>. C) Serial sections from xenografts with weight < 150mg were screened for intratubular NuMA positive cells and percentage of positive tubules was calculated. Data are represented as mean ± SD, n ≥ 3. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s009" target="_blank">S4 Table</a>. D) Immunofluorescence staining for human specific NuMA (red) and DDX4 (green). Human fetal testis sample was used as positive control. Representative images with intratubular NuMA positive and DDX4 negative cells are shown for <i>pbMOCK</i>, <i>pbNANOS3</i> and <i>pbDAZL</i> xenografts. Cells were counterstained with DAPI (blue). Scale bar: 100 μm.</p

Analysis of <i>in vitro</i> differentiated <i>pbNANOS3</i> and <i>pbDAZL</i> cells.

<p><i>A</i>) Gene expression analysis for cells differentiated on SNL-feeders using SSC medium for 5, 7, 10 or 14 days, and for cells in undifferentiated culture conditions (D0). Samples from three separate experiments were used at passage 6–8 after transfection. Values are relative quantities normalized to <i>GAPDH</i> and <i>RPLPO</i>, and represented as mean ± SD. Statistical significance was tested by two-way ANOVA, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s008" target="_blank">S3 Table</a>. B) Immunofluorescence staining of transfected cells differentiated for 14 days on SNL-feeders; PLZF (green), GFRa1 (red), NANOS3 (red) and DAZL (red). Cells were counterstained with DAPI (blue). Scale bars: 100 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s003" target="_blank">S3 Fig</a>.</p

Differentially expressed genes in <i>pbNANOS3</i> and <i>pbDAZL</i> cells in undifferentiated culture conditions.

<p>A) Differential gene expression analysis for transfected cells by mRNA sequencing. Samples from three separate transfections were used at passage 2 after transfection. Results are shown in MA-plot with log fold change (logFC) values in the <i>pbNANOS3</i> or <i>pbDAZL</i> versus the <i>pbMOCK</i> sample, plotted against average log counts per million (logCPM). Significant genes (FDR < 0.05) are marked as red: <i>pbNANOS3</i> (4 genes), <i>pbDAZL</i> (38 genes). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s007" target="_blank">S2 Table</a>. Selected differentially expressed genes for B) <i>pbNANOS3</i> and C) <i>pbDAZL</i>, with suggested functions. Values are Fragments Per Kilobase Of Exon Per Million Fragments (FPKM), and represented as mean ± SD. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s001" target="_blank">S1D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165268#pone.0165268.s002" target="_blank">S2</a> Figs.</p