Investigating the impact of vitrification on bovine ovarian tissue morphology, follicle survival, and transcriptomic signature.

PURPOSE: Ovarian tissue cryopreservation is vital for fertility preservation, yet its effect on ovarian tissue follicle survival and transcriptomic signature requires further investigation. This study delves into the effects of vitrification on tissue morphology, function, and transcriptomic changes, helping to find possibilities for vitrification protocol improvements. METHODS: Ovarian cortex from 19 bovine animals were used to conduct pre- and post-vitrification culture followed by histological assessment, immunohistochemistry, and TUNEL assay. Follicles' functionality was assessed for viability and growth within the tissue and in isolated cultures. RNA-sequencing of ovarian tissue was used to explore the transcriptomic alterations caused by vitrification. RESULTS: Follicle density, cell proliferation, and DNA damage in ovarian stroma were unaffected by vitrification. However, vitrified cultured tissue exhibited reduced follicle density of primordial/primary and antral follicles, while freshly cultured tissue manifested reduction of antral follicles. Increased stromal cell proliferation and DNA damage occurred in both groups post-culture. Isolated follicles from vitrified tissue exhibited similar viability to fresh follicles until day 4, after which the survival dropped. RNA-sequencing revealed minor effects of vitrification on transcriptomic signatures, while culture induced significant gene expression changes in both groups. The altered expression of WNT and hormonal regulation pathway genes post-vitrification suggests the molecular targets for vitrification protocol refinement. CONCLUSION: Vitrification minimally affects tissue morphology, follicle density, and transcriptomic signature post-thawing. However, culture revealed notable changes in vitrified tissue samples, including reduced follicle density, decreased isolated follicle survival, and alteration in WNT signalling and ovarian hormonal regulation pathways, highlighted them as possible limitations of the current vitrification protocol.

Induced Pluripotent Stem Cells as a Possible Approach for Exploring the Pathophysiology of Polycystic Ovary Syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine condition among women with pleiotropic sequelae possessing reproductive, metabolic, and psychological characteristics. Although the exact origin of PCOS is elusive, it is known to be a complex multigenic disorder with a genetic, epigenetic, and environmental background. However, the pathogenesis of PCOS, and the role of genetic variants in increasing the risk of the condition, are still unknown due to the lack of an appropriate study model. Since the debut of induced pluripotent stem cell (iPSC) technology, the ability of reprogrammed somatic cells to self-renew and their potential for multidirectional differentiation have made them excellent tools to study different disease mechanisms. Recently, researchers have succeeded in establishing human in vitro PCOS disease models utilizing iPSC lines from heterogeneous PCOS patient groups (iPSCPCOS). The current review sets out to summarize, for the first time, our current knowledge of the implications and challenges of iPSC technology in comprehending PCOS pathogenesis and tissue-specific disease mechanisms. Additionally, we suggest that the analysis of polygenic risk prediction based on genome-wide association studies (GWAS) could, theoretically, be utilized when creating iPSC lines as an additional research tool to identify women who are genetically susceptible to PCOS. Taken together, iPSCPCOS may provide a new paradigm for the exploration of PCOS tissue-specific disease mechanisms.

CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate.

The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms' tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Reference standards for follicular density in ovarian cortex from birth to sexual maturity.

RESEARCH QUESTION: Are age-normalized reference values for human ovarian cortical follicular density adequate for tissue quality control in fertility preservation? DESIGN: Published quantitative data on the number of follicles in samples without known ovarian pathology were converted into cortical densities to create reference values. Next, a sample cohort of 126 girls (age 1-24 years, mean ± SD 11 ± 6) with cancer, severe haematological disease or Turner syndrome were used to calculate Z-scores for cortical follicular density based on the reference values. RESULTS: No difference was observed between Z-scores in samples from untreated patients (0.3 ± 3.5, n = 30) and patients treated with (0.5 ± 2.9, n = 48) and without (0.1 ± 1.3, n = 6) alkylating chemotherapy. Z-scores were not correlated with increasing cumulative exposure to cytostatics. Nevertheless, Z-scores in young treated patients (0-2 years -2.1 ± 3.1, n = 10, P = 0.04) were significantly lower than Z-scores in older treated patients (11-19 years, 2 ± 1.9, n = 15). Samples from patients with Turner syndrome differed significantly from samples from untreated patients (-5.2 ± 5.1, n = 24, P = 0.003), and a Z-score of -1.7 was identified as a cut-off showing good diagnostic value for identification of patients with Turner syndrome with reduced ovarian reserve. When this cut-off was applied to other patients, analysis showed that those with indications for reduced ovarian reserve (n = 15) were significantly younger (5.9 ± 4.2 versus 10.7 ± 5.9 years, P = 0.004) and, when untreated, more often had non-malignant haematologic diseases compared with those with normal ovarian reserve (n = 24, 100% versus 19%, P = 0.009). CONCLUSION: Z-scores allow the estimation of genetic- and treatment-related effects on follicular density in cortical tissue from young patients stored for fertility preservation. Understanding the quality of cryopreserved tissue facilitates its use during patient counselling. More research is needed regarding the cytostatic effects found in this study.

Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.

Deciphering the Transcriptional Landscape of Human Pluripotent Stem Cell-Derived GnRH Neurons: The Role of Wnt Signaling in Patterning the Neural Fate.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons lay the foundation for human development and reproduction; however, the critical cell populations and the entangled mechanisms underlying the development of human GnRH neurons remain poorly understood. Here, by using our established human pluripotent stem cell-derived GnRH neuron model, we decoded the cellular heterogeneity and differentiation trajectories at the single-cell level. We found that a glutamatergic neuron population, which generated together with GnRH neurons, showed similar transcriptomic properties with olfactory sensory neuron and provided the migratory path for GnRH neurons. Through trajectory analysis, we identified a specific gene module activated along the GnRH neuron differentiation lineage, and we examined one of the transcription factors, DLX5, expression in human fetal GnRH neurons. Furthermore, we found that Wnt inhibition could increase DLX5 expression and improve the GnRH neuron differentiation efficiency through promoting neurogenesis and switching the differentiation fates of neural progenitors into glutamatergic neurons/GnRH neurons. Our research comprehensively reveals the dynamic cell population transition and gene regulatory network during GnRH neuron differentiation.

FGF8-FGFR1 signaling regulates human GnRH neuron differentiation in a time- and dose-dependent manner.

Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time- and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (P<0.05, Rs=0.49). However, cells treated with 100 ng/ml FGF8 for 2 days induced the expression of genes, such as FOXG1, ETV5 and SPRY2, and continued FGF8 treatment induced the dynamic expression of several other genes. Moreover, during exposure to FGF8, FGFR1 localized to the cell surface and its specific inhibition with the FGFR1 inhibitor PD166866 reduced expression of GNRH1 (P<0.05). In neurons, FGFR1 also localized to the nucleus. Our results suggest that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny. This article has an associated First Person interview with the first author of the paper.

DUX4 is a multifunctional factor priming human embryonic genome activation.

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

Proteome-wide and matrisome-specific atlas of the human ovary computes fertility biomarker candidates and open the way for precision oncofertility.

Our modern era is witnessing an increasing infertility rate worldwide. Although some of the causes can be attributed to our modern lifestyle (e.g., persistent organic pollutants, late pregnancy), our knowledge of the human ovarian tissue has remained limited and insufficient to reverse the infertility statistics. Indeed, all efforts have been focused on the endocrine and cellular function in support of the cell theory that dates back to the 18th century, while the human ovarian matrisome is still under-described. Hereby, we unveil the extracellular side of the story during different periods of the ovary life, demonstrating that follicle survival and development, and ultimately fertility, would not be possible without its involvement. We examined the human ovarian matrisome and described its remodeling from prepuberty until menopause, creating the first ovarian proteomic codex. Here, we confidently identified and quantified 98 matrisome proteins present in the three ovary groups. Among them, 26 were expressed differently among age groups, delineating a peculiar matrisomal fingerprint at each stage. Such proteins could be potential biomarkers phenotyping ovarian ECM at each age phase of female reproductive life. Beyond proteomics, our study presents a unique approach to understanding the data and depicting the spatiotemporal ECM-intracellular signaling networks and remodeling with age through imaging, advanced text-mining based on natural language processing technology, machine learning, and data sonification. Our findings provide essential context for healthy ovarian physiology, identifying and characterizing disease states, and recapitulating physiological tissues or development in vitro. This comprehensive proteomics analysis represents the ovarian proteomic codex and contributes to an improved understanding of the critical roles that ECM plays throughout the ovarian life span.

A blueprint of the topology and mechanics of the human ovary for next-generation bioengineering and diagnosis.

Although the first dissection of the human ovary dates back to the 17th century, the biophysical characteristics of the ovarian cell microenvironment are still poorly understood. However, this information is vital to deciphering cellular processes such as proliferation, morphology and differentiation, as well as pathologies like tumor progression, as demonstrated in other biological tissues. Here, we provide the first readout of human ovarian fiber morphology, interstitial and perifollicular fiber orientation, pore geometry, topography and surface roughness, and elastic and viscoelastic properties. By determining differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, we unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. While these data enable to design of more biomimetic scaffolds for the tissue-engineered ovary, our analysis pipeline is applicable for the characterization of other organs in physiological or pathological states to reveal their biophysical markers or design their bioinspired analogs.

Small RNA expression and miRNA modification dynamics in human oocytes and early embryos.

Small noncoding RNAs (sRNAs) play important roles during the oocyte-to-embryo transition (OET), when the maternal phenotype is reprogrammed and the embryo genome is gradually activated. The transcriptional program driving early human development has been studied with the focus mainly on protein-coding RNAs, and expression dynamics of sRNAs remain largely unexplored. We profiled sRNAs in human oocytes and early embryos using an RNA-sequencing (RNA-seq) method suitable for low inputs of material. We show that OET in humans is temporally coupled with the transition from predominant expression of oocyte short piRNAs (os-piRNAs) in oocytes, to activation of microRNA (miRNA) expression in cleavage stage embryos. Additionally, 3' mono- and oligoadenylation of miRNAs is markedly increased in zygotes. We hypothesize that this may modulate the function or stability of maternal miRNAs, some of which are retained throughout the first cell divisions in embryos. This study is the first of its kind elucidating the dynamics of sRNA expression and miRNA modification along a continuous trajectory of early human development and provides a valuable data set for in-depth interpretative analyses.

Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells.

Gonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.

Complement in Human Pre-implantation Embryos: Attack and Defense.

It is essential for early human life that mucosal immunological responses to developing embryos are tightly regulated. An imbalance of the complement system is a common feature of pregnancy complications. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus, far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation against developing human embryos takes place already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products on healthy developing embryos, which highlights the need for strict complement regulation. We show that embryos express complement membrane inhibitors and bind soluble regulators. These findings show that mucosal complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing revealed cellular expression of complement activators. This shows that the embryonic cells themselves have the capacity to express and activate C3 and C5. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and furthermore may impact cellular homeostasis and differentiation at the earliest stages of life.

MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression.

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1-expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.

Demographic and evolutionary trends in ovarian function and aging.

BACKGROUND: The human female reproductive lifespan is regulated by the dynamics of ovarian function, which in turn is influenced by several factors: from the basic molecular biological mechanisms governing folliculogenesis, to environmental and lifestyle factors affecting the ovarian reserve between conception and menopause. From a broader point of view, global and regional demographic trends play an additional important role in shaping the female reproductive lifespan, and finally, influences on an evolutionary scale have led to the reproductive senescence that precedes somatic senescence in humans. OBJECTIVE AND RATIONALE: The narrative review covers reproductive medicine, by integrating the molecular mechanisms of ovarian function and aging with short-term demographic and long-term evolutionary trends. SEARCH METHODS: PubMed and Google Scholar searches were performed with relevant keywords (menopause, folliculogenesis, reproductive aging, reproductive lifespan and life history theory). The reviewed articles and their references were restricted to those written in English. OUTCOMES: We discuss and summarize the rapidly accumulating information from large-scale population-based and single-reproductive-cell genomic studies, their constraints and advantages in the context of female reproductive aging as well as their possible evolutionary significance on the life history trajectory from foetal-stage folliculogenesis until cessation of ovarian function in menopause. The relevant environmental and lifestyle factors and demographic trends are also discussed in the framework of predominant evolutionary hypotheses explaining the origin and maintenance of menopause. WIDER IMPLICATIONS: The high speed at which new data are generated has so far raised more questions than it has provided solid answers and has been paralleled by a lack of satisfactory interpretations of the findings in the context of human life history theory. Therefore, the recent flood of data could offer an unprecedented tool for future research to possibly confirm or rewrite human evolutionary reproductive history, at the same time providing novel grounds for patient counselling and family planning strategies.

Hyperglycosylated hCG activates LH/hCG-receptor with lower activity than hCG.

While human chorionic gonadotropin (hCG) appears to have an essential role in early pregnancy, it is controversial whether the hyperglycosylated form of hCG (hCG-h), which is the major hCG isoform during the first 4-5 weeks of pregnancy, is able to activate LH/hCG receptor (LHCGR). To address this, we utilized different extensively characterized hCG and hCGß reference reagents, cell culture- and urine-derived hCG-h preparations, and an in vitro reporter system for LHCGR activation. The WHO hCG reference reagent (99/688) was found to activate LHCGR with an EC50-value of 3.3 ±â€¯0.6 pmol/L (n = 9). All three studied hCG-h preparations were also able to activate LHCGR, but with a lower potency (EC50-values between 7.1 ±â€¯0.5 and 14 ±â€¯3 pmol/L, n = 5-11, for all P < 0.05 as compared to the hCG reference). The activities of commercial urinary hCG (Pregnyl) and recombinant hCG (Ovitrelle) preparations were intermediate between those of the hCG reference and the hCG-h. These results strongly suggest that the hCG-h is functionally similar to hCG, although it has lower potency for LHCGR activation. Whether this explains the reduced proportion of hCG-h to hCG reported in patients developing early onset pre-eclampsia or those having early pregnancy loss remains to be determined.

rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology.

Background: Assisted reproductive technology (ART) has been associated with low birth weight of fresh embryo transfer (FRESH) derived and increased birth weight of frozen embryo transfer (FET)-derived newborns. Owing to that, we focused on imprinted insulin-like growth factor 2 (IGF2)/H19 locus known to be important for normal growth. This locus is regulated by H19 imprinting control region (ICR) with seven binding sites for the methylation-sensitive zinc finger regulatory protein (CTCF). A polymorphism rs10732516 G/A in the sixth binding site for CTCF, associates with a genotype-specific trend to the DNA methylation. Due to this association, 62 couples with singleton pregnancies derived from FRESH (44 IVF/18 ICSI), 24 couples from FET (15 IVF/9 ICSI), and 157 couples with spontaneously conceived pregnancies as controls were recruited in Finland and Estonia for genotype-specific examination. DNA methylation levels at the H19 ICR, H19 DMR, and long interspersed nuclear elements in placental tissue were explored by MassARRAY EpiTYPER (n = 122). Allele-specific changes in the methylation level of H19 ICR in placental tissue (n = 26) and white blood cells (WBC, n = 8) were examined by bisulfite sequencing. Newborns' (n = 243) anthropometrics was analyzed by using international growth standards. Results: A consistent trend of genotype-specific decreased methylation level was observed in paternal allele of rs10732516 paternal A/maternal G genotype, but not in paternal G/maternal A genotype, at H19 ICR in ART placentas. This hypomethylation was not detected in WBCs. Also genotype-specific differences in FRESH-derived newborns' birth weight and head circumference were observed (P = 0.04, P = 0.004, respectively): FRESH-derived newborns with G/G genotype were heavier (P = 0.04) and had larger head circumference (P = 0.002) compared to newborns with A/A genotype. Also, the placental weight and birth weight of controls, FRESH- and FET-derived newborns differed significantly in rs10732516 A/A genotype (P = 0.024, P = 0.006, respectively): the placentas and newborns of FET-derived pregnancies were heavier compared to FRESH-derived pregnancies (P = 0.02, P = 0.004, respectively). Conclusions: The observed DNA methylation changes together with the phenotypic findings suggest that rs10732516 polymorphism associates with the effects of ART in a parent-of-origin manner. Therefore, this polymorphism should be considered when the effects of environmental factors on embryonic development are studied.

The Role of Sequential BMP Signaling in Directing Human Embryonic Stem Cells to Bipotential Gonadal Cells.

Context: Human gonads arise as a pair of epithelial ridges on the surface of intermediate mesoderm (IM)-derived mesonephros. Toxic environmental factors and mutations in various genes are known to disturb normal gonadal development, but because of a lack of suitable in vitro models, detailed studies characterizing the molecular basis of the observed defects have not been performed. Objective: To establish an in vitro method for studying differentiation of bipotential gonadal progenitors by using human embryonic stem cells (hESCs) and to investigate the role of bone morphogenetic protein (BMP) in gonadal differentiation. Design: We tested 17 protocols using activin A, CHIR-99021, and varying durations of BMP-7 and the BMP inhibitor dorsomorphin. Activation of activin A, WNT, and BMP pathways was optimized to induce differentiation. Setting: Academic research laboratory. Main Outcomes Measures: Cell differentiation, gene expression, and flow cytometry. Results: The two most efficient protocols consistently upregulated IM markers LHX1, PAX2, and OSR1 at days 2 to 4 and bipotential gonadal markers EMX2, GATA4, WT1, and LHX9 at day 8 of culture. The outcome depended on the combination of the duration, concentration, and type of BMP activation and the length of WNT signaling. Adjusting any of the parameters substantially affected the requirements for other parameters. Conclusions: We have established a reproducible protocol for directed differentiation of hESCs into bipotential gonadal cells. The protocol can be used to model early gonadal development in humans and allows further differentiation to mature gonadal somatic cells.

Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-ß/activin/nodal signalling inhibition.

Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 (BMP4) and/or inhibitors of fibroblast growth factor 2 (FGF2) and the transforming growth factor beta (TGF-ß)/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin (HCG) and its hyperglycosylated form (HCG-H) were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes (CGA, CGB and LGALS16), induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-ß/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy.