Reproductive health underpins overall health, and thus, research in reproductive science and medicine is essential. This requires a pipeline of trained scientists and clinicians, which is challenging given the relatively small size of the field. Educational programs outside the traditional doctorate or medical degrees are needed to generate and maintain a well-trained reproductive science and medicine workforce. Master's programs have gained traction as a viable way for students to receive educational value relative to their career goals. Our hypothesis is master's degree programs in the fundamental, applied, and allied health reproductive fields broadens the workforce and increases impact. An internet web search identified 73 programs that conferred an MS degree in the areas of animal science, clinical/embryology, and reproductive health/biology. These programs are spread across the globe in Europe (47%), North America (23%), Asia (14%), South America (7%), Oceania (5%), and Africa (4%). To evaluate global exemplars, we profiled the mission and structure, curriculum, and program impact of two established master's degree programs: the Master of Science in Reproductive Science and Medicine at Northwestern University in the United States and the Biology and Technology of Reproduction in Mammals at the University of Murcia in Spain. Elements of infrastructure and support, program connectivity, and alumni networks were analyzed for their role in the success of the programs. These two programs have formally trained >375 individuals, demonstrating master's degree programs in reproductive science are an important educational mechanism. The educational best practices shared here serve as templates for expanding training programs worldwide.

Summary Sentence

MS programs in reproductive science are a critical and growing training pipeline in our field as evidenced by a survey of global programs as well as benchmarking studies of the Master of Science in Reproductive Science and Medicine program (MS-RSM) at Northwestern University in the United States and the Biology and Technology of Reproduction in Mammals at the University of Murcia in Spain.

Precocious puberty, as a common pediatric endocrine disease, can be divided into central precocious puberty and peripheral precocious puberty, even though most cases of precocious puberty are diagnosed as central precocious puberty. According to its etiology, central precocious puberty can be further divided into organic and idiopathic central precocious puberty. However, the mechanisms of idiopathic central precocious puberty have not yet been fully elucidated. Currently, four genes, including the kisspeptin gene, the kisspeptin receptor gene, the makorin ring finger protein 3, and the delta-like noncanonical Notch ligand 1, have been implicated in central precocious puberty cases, of which delta-like noncanonical Notch ligand 1 has been determined to represent a key, recently found central precocious puberty-related gene. In this review, we will not only highlight the latest discoveries on the relationship between the delta-like noncanonical Notch ligand 1 system and central precocious puberty but also explore the involvement of the system as well as the Notch signaling pathway in central precocious puberty occurrence.

Summary Sentence

Highlights the latest discoveries on the relationship between the Delta-like noncanonical Notch ligand 1 system and central precocious puberty (CPP) and explores the involvement of the system as well as the Notch signaling pathway in CPP occurrence.

In the recent years a special attention has been given to a major health concern namely to male infertility, defined as the inability to conceive after 12 months of regular unprotected sexual intercourse, taken into account the statistics that highlight that sperm counts have dropped by 50–60% in recent decades. According to the WHO, infertility affects approximately 9% of couples globally, and the male factor is believed to be present in roughly 50% of cases, with exclusive responsibility in 30%. The aim of this article is to present an evidence-based approach for diagnosing male infertility that includes finding new solutions for diagnosis and critical outcomes, retrieving up-to-date studies and existing guidelines. The diverse factors that induce male infertility generated in a vast amount of data that needed to be analyzed by a clinician before a decision could be made for each individual. Modern medicine faces numerous obstacles as a result of the massive amount of data generated by the molecular biology discipline. To address complex clinical problems, vast data must be collected, analyzed, and used, which can be very challenging. The use of artificial intelligence (AI) methods to create a decision support system can help predict the diagnosis and guide treatment for infertile men, based on analysis of different data as environmental and lifestyle, clinical (sperm count, morphology, hormone testing, karyotype, etc.), and “omics” bigdata. Ultimately, the development of AI algorithms will assist clinicians in formulating diagnosis, making treatment decisions, and predicting outcomes for assisted reproduction techniques.

Summary Sentence

The article intends to present an evidence-based approach for the diagnosis of male infertility. These involve many molecular aspects and the necessity of artificial intelligence techniques for diagnosis prediction and treatment guidance.

Graphical Abstract

Viviparity and the development of a placenta are two of the major reasons for the success of the mammals in colonizing all habitats, both terrestrial and aquatic. The placenta is an apposition of fetal to maternal tissue which serves two main, but competing functions: to maximize oxygen transfer and the acquisition of nutrients from the mother, but to minimize immunological rejection by the maternal immune system. This has resulted in the evolution of four main types differing in the degree of loss of the maternal uterine epithelial (UE) barrier: epitheliochorial, synepitheliochorial, endotheliochorial, and hemochorial, all providing a successful safe balance between the needs of mother and fetus. Epitheliochorial is the least invasive, a simple apposition and microvillar interdigitation of the apices of uterine epithelium and trophoblast. It is suggested to have evolved as a response to the increase in the size of the animal to provide a sufficiently long gestation to produce a single altricial (run/swim-soon-as-born) neonate as in the Cetartiodactyla. The mother needs to have good control of the fetal demands so the UE barrier is maintained. However, in the synepitheliochorial placenta, characteristic of all ruminants, the fetus has evolved a means of increasing, or at least maintaining, demand without the need for invasion. This has been achieved by the development of the trophoblast binucleate cell which, uniquely, can fuse with a UE cell to form fetomaternal hybrid tissue. This can maintain some maternal barrier function but also deliver fetally synthesized immunomodulatory and metabolic messages to the maternal circulation. This review provides the evidence for this remarkable evolutionary step and also considers an alternative explanation for the formation of the structure of the ruminant placenta.

Using somatic cell nuclear transfer for the generation of cloned and transgenic animals bears the risk of transmission of viruses, either by the oocyte or by the introduced donor cell. There is evidence that the zona pellucida (ZP) surrounding the oocyte prevents virus infection; however, virus infections despite intact ZP were reported. Furthermore, the protective ZP has to be penetrated to place the somatic cell in the oocyte's perivitelline space during SCNT. Transmission of viruses also represents a severe problem during in vitro fertilization (IVF). Genetically modified and IVF-produced pigs serve as an important biomedical model for numerous diseases and it is important to evaluate whether infections of the model animals can falsify the research data. Of special significance is this topic in the case of xenotransplantation using genetically modified pigs as donor animals, because transmission of porcine viruses may be harmful to the human recipient. This was repeatedly demonstrated in preclinical pig to non-human primate trials. Therefore, donor pigs, oocytes used for SCNT, and genetically modified donor cells should be screened for potentially zoonotic viruses when creating genetically modified pigs designed for xenotransplantation.

Summary Sentence

To avoid transmission of viruses by somatic cell nuclear transfer, a screening of the donor animals and the derived oocytes is required not only when creating genetically modified pigs for xenotransplantation, but also in other animal models of human disease.

PETER PAN (PPAN), located to nucleoli and mitochondria, is a member of the Brix domain protein family, involved in rRNA processing through its rRNA binding motif and mitochondrial apoptosis by protecting mitochondria structure and suppressing basal autophagic flux. Ppan is important for cell proliferation and viability, and mutation of Ppan in Drosophila caused larval lethality and oogenesis failure. Yet, its role in mammalian reproduction remains unclear. In this study, we explored the function of Ppan in oocyte maturation and early embryogenesis using conditional knockout mouse model. Deficiency of maternal Ppan significantly downregulated the expression level of 5.8S rRNA, 18S rRNA, and 28S rRNA, though it had no effect on oocyte maturation or preimplantation embryo development. However, depletion of both maternal and zygotic Ppan blocked embryonic development at morula stage. Similar phenotype was obtained when only zygotic Ppan was depleted. We further identified no DNA binding activity of PPAN in mouse embryonic stem cells, and depletion of Ppan had minimum impact on transcriptome but decreased expression of 5.8S rRNA, 18S rRNA, and 28S rRNA nevertheless. Our findings demonstrate that Ppan is indispensable for early embryogenesis in mice.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride/bicarbonate ion channel in epithelial cells. Mutations in CFTR cause cystic fibrosis, a disease characterized by thickened mucus secretions and is associated with subfertility and infertility. CFTR function has been well characterized in vitro and in vivo in airway and other epithelia studies. However, little is known about CFTR function in the cervix in health and its contribution to cyclic regulation of fertility from endocervical mucus changes. Contributing to this research gap is the lack of information on the effect of sex steroid hormones on CFTR expression in cervical epithelial cells across the menstrual cycle. Herein, we demonstrate the hormonal regulation of CFTR expression in endocervical cells both in vitro and in vivo, and that conditionally reprogrammed endocervical epithelial cells can be used to interrogate CFTR ion channel function. CFTR activity was demonstrated in vitro using electrophysiological methods and functionally inhibited by the CFTR-specific inhibitors inh-172 and GlyH-101. We also report that CFTR expression is increased by estradiol in the macaque cervix both in vitro and in vivo in Rhesus macaques treated with artificial menstrual cycles. Estrogen upregulation of CFTR is blocked in vivo by cotreatment with progesterone. Our findings provide the most comprehensive evidence to date that steroid hormones drive changes in CFTR expression. These data are integral to understanding the role of CFTR as a fertility regulator in the endocervix.

Summary Sentence

Cystic fibrosis transmembrane conductance regulator is hormonally regulated and functional in the macaque endocervix.

AbstractThe myometrium undergoes progressive tissue remodeling from early to late pregnancy to support fetal growth and transitions to the contractile phase to deliver a baby at term. Much of our effort has been focused on understanding the functional role of myometrial smooth muscle cells, but the role of extracellular matrix is not clear. This study was aimed to demonstrate the expression profile of sub-sets of genes involved in the synthesis, processing, and assembly of collagen and elastic fibers, their structural remodeling during pregnancy, and hormonal regulation. Myometrial tissues were isolated from non-pregnant and pregnant mice to analyze gene expression and protein levels of components of collagen and elastic fibers. Second harmonic generation imaging was used to examine the morphology of collagen and elastic fibers. Gene and protein expressions of collagen and elastin were induced very early in pregnancy. Further, the gene expressions of some of the factors involved in the synthesis, processing, and assembly of collagen and elastic fibers were differentially expressed in the pregnant mouse myometrium. Our imaging analysis demonstrated that the collagen and elastic fibers undergo structural reorganization from early to late pregnancy. Collagen and elastin were differentially induced in response to estrogen and progesterone in the myometrium of ovariectomized mice. Collagen was induced by both estrogen and progesterone. By contrast, estrogen induced elastin, but progesterone suppressed its expression. The current study suggests progressive extracellular matrix remodeling and its potential role in the myometrial tissue mechanical function during pregnancy and parturition.

Graphical Abstract

Summary Sentence

The collagen and elastic fibers undergo structural reorganization from early to late pregnancy and the expression of both collagen and elastin is under the influence of steroid hormones estrogen and progesterone.

Prssly (Protease, serine-like, Chr Y) and Teyorf1 (Testis expressed, chromosome Y open reading frame 1) are two acquired single-copy genes located on the distal tip of the non-pairing short arm of the mouse Y chromosome adjacent to telomeric sequence. Both genes lack X chromosome-linked homologues and are expressed in testicular germ cells. We first performed analysis of Prssly and Teyorf1 genomic sequences and demonstrated that previously reported Prssly sequence is erroneous and the true Prssly sequence is longer and encodes a larger protein than previously estimated. We also confirmed that both genes encode pseudogenes that are not expressed in testes. Next, using CRISPR/Cas9 genome targeting, we generated Prssly and Teyorf1 knockout (KO) mice and characterized their phenotype. To create Prssly KO mice, we targeted the conserved exon 5 encoding a trypsin domain typical for serine proteases. The targeting was successful and resulted in a frame shift mutation that introduced a premature stop codon, with the Prssly KO males retaining only residual transcript expression in testes. The Teyorf1 targeting removed the entire open reading frame of the gene, which resulted in no transcript expression in KO males. Both Prssly KO and Teyorf1 KO males were fertile and had normal testis size and normal sperm number, motility, and morphology. Our findings show that Prssly and Teyorf1 transcripts with potential to encode proteins are dispensable for male fertility.

Summary Sentence

CRISPR/Cas9 generated Prssly and Teyorf1 knockout mice are fertile and display normal spermatogenic features.

In oocytes, mRNA decay is essential for maturation and subsequent events, such as maternal-zygotic transition, zygotic genomic activation, and embryo development. Reversible N6-methyladenosine RNA methylation directly regulates transcription, pre-mRNA splicing, mRNA export, mRNA stability, and translation. Here, we identified that downregulation of N6-methyladenosine modification by microinjecting a methyltransferase-like 3 (Mettl3)-specific small interfering RNA into mouse germinal vesicle oocytes led to defects in meiotic spindles and the first polar body extrusion during maturation in vitro. By further quantitative real-time polymerase chain reaction and Poly(A)-tail assay analysis, we found that N6-methyladenosine methylation mainly acts by reducing deadenylation of mRNAs mediated by the carbon catabolite repression 4-negative on TATA less system, thereby causing mRNA accumulation in oocytes. Meanwhile, transcriptome analysis of germinal vesicle oocytes revealed the downregulation of transcripts of several genes encoding ribosomal subunits proteins in the Mettl3 small interfering RNA-treated group, suggesting that N6-methyladenosine modification might affect translation. Together, our results indicate that RNA methylation accelerates mRNA decay, confirming the critical role of RNA clearance in oocyte maturation.

Exogenous estrogen have shown their feminization abilities during the specific sex differentiation period in several reptiles. However, the specific regulatory mechanism and downstream regulatory genes of estrogen remain elusive. In the present study, 17β-estradiol (E₂), as well as drugs of specific antagonists and/or agonists of estrogen receptors, were employed to figure out the molecular pathway involved in the E₂-induced feminization in Chinese soft-shelled turtles, an important aquaculture species in China. E₂ treatment led to typical female characteristics in the gonads of ZZ individuals, including thickened outer cortex containing a number of germ cells and degenerated medullary cords, as well as the disappearance of male marker SOX9, and the ectopic expression of ovarian regulator FOXL2 at the embryonic developmental stage 27 and 1 month after hatching. The specific ESR1 antagonist or a combination of three estrogen receptor antagonists could block the sex reversal of ZZ individuals induced by estrogen. In addition, specific activation of ESR1 by agonist also led to the feminization of ZZ gonads, which was similar to the effect of estrogen treatment. Furthermore, transcriptome data showed that the expression level of FOXL2 was significantly upregulated, whereas mRNA levels of DMRT1, SOX9, and AMH were downregulated after estrogen treatment. Taken together, our results indicated that E₂ induced the feminization of ZZ Chinese soft-shelled turtles via ESR1, and decrease of male genes DMRT1, SOX9, and AMH and increase of ovarian development regulator FOXL2 might be responsible for the initiation of E₂-induced feminization.

Graphical Abstract

Fanconi anemia complementation group B (FANCB) protein is a major component of the Fanconi anemia (FA) core complex and plays an important role in hematopoiesis and germ cell development. Deletion of Fancb gene causes the defect of primordial germ cell (PGC) development and infertility in male mice. However, it remains unknown whether Fancb is required for female germ cell development. In this study, we found that the fertility of Fancb knockout male mice in C57/ICR mixed backgrounds was not affected. Female Fancb^–/– mice were obtained by crossing Fancb^+/– females with Fancb–^/Y males. The number of PGCs was dramatically decreased in Fancb^–/– females. Very few oocytes were observed after birth and the primordial follicle pool was completely depleted at 6 weeks of age in Fancb^–/– females. However, the remained oocytes from Fancb^–/– mice were normal in fertilization and embryonic development from 2-cell to the blastocyst stage. We also found that Fancb and Fancl double-knockout males were also fertile and the number of sperm in epididymis was not reduced as compared to that of Fancb^–/– and Fancl^–/– single-knockout mice. Taken together, these results showed that Fancb is also essential for female germ cell development. Inactivation of Fancb causes massive germ cell loss and infertility in adult females. We also found that Fancb and Fancl do not act synergistically in regulating germ cell development.

Summary Sentence

Fancb plays an essential role in the maintenance of germ cell development in female gonads and is a potentially causative gene for premature ovarian insufficiency.

Graphical Abstract

Estradiol-17β has been shown to promote primordial follicle formation and to involve bone morphogenetic protein 2 (BMP2) as a downstream effector to promote primordial follicle in hamsters. However, the molecular mechanism whereby these factors regulate ovarian somatic cells to pre-granulosa cells transition leading to primordial follicle formation remains unclear. The objective of this study was to determine whether BMP2 and/or estradiol-17β would regulate the expression of specific ovarian transcriptome during pre-granulosa cells transition and primordial follicle formation in the mouse ovary. BMP2 mRNA level increased during the period of primordial follicle formation with the concurrent presence of BMP2 protein in ovarian somatic cells. Estradiol-17β but not BMP2 exposure led to increased expression of ovarian BMP2 messenger RNA (mRNA), and the effect of estradiol-17β could not be suppressed by 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride (LDN) 193189. BMP2 or estradiol-17β stimulated primordial follicle formation without inducing apoptosis. Ribonucleic acid-sequence analysis (RNA-seq) of ovaries exposed to exogenous BMP2 or estradiol-17β revealed differential expression of several thousand genes. Most of the differentially expressed genes, which were common between BMP2 or estradiol-17β treatment demonstrated concordant changes, suggesting that estradiol-17β and BMP2 affected the same set of genes during primordial follicle formation. Further, we have identified that estradiol-17β, in cooperation with BMP2, could affect the expression of three major transcription factors, GATA binding protein 2, GATA binding protein 4 and Early growth response 2, and one serine protease, hepsin, in pre-granulosa cells during primordial follicle formation. Taken together, results of this study suggest that estradiol-17β and BMP2 may regulate ovarian gene expression that promote somatic cells to pre-granulosa cells transition and primordial follicle formation in the mouse ovary.

Summary Sentence

BMP2 and estradiol-17β may regulate the transition of somatic cells to pre-granulosa cells, leading to the formation of primordial follicles in the murine ovary by upregulating key ovarian transcriptome.

The anti-Müllerian hormone (AMH) produced by the granulosa cells of growing follicles is critical for folliculogenesis and is clinically used as a diagnostic and prognostic marker of female fertility. Previous studies report that AMH-pretreatment in mice creates a pool of quiescent follicles that are released following superovulation, resulting in an increased number of ovulated oocytes. However, the quality and developmental competency of oocytes derived from AMH-induced accumulated follicles as well as the effect of AMH treatment on live birth are not known. This study reports that AMH priming positively affects oocyte maturation and early embryonic development culminating in higher number of live births. Our results show that AMH treatment results in good-quality oocytes with greater developmental competence that enhances embryonic development resulting in blastocysts with higher gene expression. The transcriptomic analysis of oocytes from AMH-primed mice compared with those of control mice reveal that AMH upregulates a large number of genes and pathways associated with oocyte quality and embryonic development. Mitochondrial function is the most affected pathway by AMH priming, which is supported by more abundant active mitochondria, mitochondrial DNA content and adenosine triphosphate levels in oocytes and embryos isolated from AMH-primed animals compared with control animals. These studies for the first time provide an insight into the overall impact of AMH on female fertility and highlight the critical knowledge necessary to develop AMH as a therapeutic option to improve female fertility.

Summary Sentence

Anti-Müllerian hormone treatment increases number of ovulated oocytes and, positively affects oocyte quality and early embryonic development resulting in higher number of live births.

During the peri-implantation period of pregnancy, the trophectoderm of pig conceptuses utilize glucose via multiple biosynthetic pathways to support elongation and implantation, resulting in limited availability of pyruvate for metabolism via the TCA cycle. Therefore, we hypothesized that porcine trophectoderm cells replenish tricarboxylic acid (TCA) cycle intermediates via a process known as anaplerosis and that trophectoderm cells convert glutamine to α-ketoglutarate, a TCA cycle intermediate, through glutaminolysis. Results demonstrate: (1) that expression of glutaminase (GLS) increases in trophectoderm and glutamine synthetase (GLUL) increases in extra-embryonic endoderm of conceptuses, suggesting that extra-embryonic endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and (2) that expression of glutamate dehydrogenase 1 (GLUD1) decreases and expression of aminotransferases including PSAT1 increase in trophectoderm, suggesting that glutaminolysis occurs in the trophectoderm through the GLS-aminotransferase pathway during the peri-implantation period. We then incubated porcine conceptuses with ¹³C-glutamine in the presence or absence of glucose in the culture media and then monitored the movement of glutamine-derived carbons through metabolic intermediates within glutaminolysis and the TCA cycle. The ¹³C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of ¹³C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.

Summary Sentence

The trophectoderm cells of pigs metabolize glucose through glycolytic biosynthetic pathways and utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux during the peri-implantation period.

In the early stage of pregnancy, hypoxia in the placenta is of great significance to the migration and invasion of trophoblasts. In addition, changes to the polarity and activity of macrophages can affect embryo implantation, trophoblast migration and invasion, and vascular remodeling by affecting cytokine secretion. However, the mechanism of the effects of hypoxic conditions in the placenta on trophoblasts remains unknown. We used gene knockdown on macrophages, and drug treatment on trophoblasts, and cultured them under hypoxic and normoxic conditions. The cells were then subjected to wound-healing assays, Transwell cell invasion experiments, quantitative real-time reverse transcription Polymerase Chain Reaction (PCR), western blotting, and immunofluorescence. The polarization of macrophages in each group, the migration and invasion ability of trophoblasts, and changes to the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were detected. Hypoxic conditions induce M2 polarization of macrophages. The conditioned medium from macrophages under hypoxic conditions increased the migration and invasion of trophoblasts and enhanced the levels of phosphorylated (p)-PI3K and p-AKT in trophoblasts. After C-C motif chemokine ligand 5 knockdown in macrophages, the ability of conditioned medium from macrophages cultured under hypoxic conditions to promote the migration and invasion of trophoblasts was weakened significantly. The use of PI3K/AKT signaling pathway agonists could reverse the attenuation effect caused by C-C motif chemokine ligand 5 knockdown.

Summary sentence

Hypoxic conditions induced the polarization of M2 macrophages and their secretion of C-C motif chemokine ligand 5, thereby activating the PI3K/AKT signaling pathway in trophoblasts, ultimately leading to enhanced trophoblast migration and invasion.

Graphical Abstract

The hypoxic environment of the placenta in early pregnancy might play a role in the mechanism of macrophage polarization, in which hypoxic conditions can induce macrophages to polarize to the M2 type and stimulate the secretion of CCL5. This will further promote the migration and invasion of trophoblast cells. At the same time, CCL5 derived from macrophages is involved in the activation of the PI3K/AKT signaling pathway in trophoblasts under hypoxic conditions.

Fetal growth restriction (FGR) describes a fetus which has not achieved its genetic growth potential; it is closely linked to placental dysfunction and uteroplacental hypoxia. Estrogen-related receptor gamma (ESRRG) is regulated by hypoxia and is highly expressed in the placenta. We hypothesized ESRRG is a regulator of hypoxia-mediated placental dysfunction in FGR pregnancies. Placentas were collected from women delivering appropriate for gestational age (AGA; n = 14) or FGR (n = 14) infants. Placental explants (n = 15) from uncomplicated pregnancies were cultured for up to 4 days in 21% or 1% O₂, or with 200 µM cobalt chloride (CoCl₂), or treated with the ESRRG agonists DY131 under different oxygen concentrations. RT-PCR, Western blotting, and immunochemistry were used to assess mRNA and protein levels of ESRRG and its localization in placental tissue from FGR or AGA pregnancies, and in cultured placental explants. ESRRG mRNA and protein expression were significantly reduced in FGR placentas, as was mRNA expression of the downstream targets of ESRRG, hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2), and cytochrome P-450 (CYP19A1.1). Hypoxia-inducible factor 1-alpha protein localized to the nuclei of the cytotrophoblasts and stromal cells in the explants exposed to CoCl₂ or 1% O₂. Both hypoxia and CoCl₂ treatment decreased ESRRG and its downstream genes' mRNA expression, but not ESRRG protein expression. DY131 increased the expression of ESRRG signaling pathways and prevented abnormal cell turnover induced by hypoxia. These data show that placental ESRRG is hypoxia-sensitive and altered ESRRG-mediated signaling may contribute to hypoxia-induced placental dysfunction in FGR. Furthermore, DY131 could be used as a novel therapeutic approach for the treatment of placental dysfunction.

The endocrine disruptive effects of bisphenol A (BPA) and brominated flame retardants (BDE-47) have led to restrictions on their use and increased the pressure to identify safe replacements for these chemicals. Although there is evidence that some of these alternatives may be toxic to spermatogonial and Leydig cells, little is known about the toxicity of emerging replacements on Sertoli cells. We used high-content imaging to compare the effects of legacy chemicals, BPA and BDE-47, to their corresponding replacements. TM4 Sertoli cells were exposed for 48 h to each chemical (0.001–100 µM) followed by cytotoxicity and phenotypic endpoint assessment. The benchmark concentration potency ranking for bisphenols based on cytotoxicity was BPTMC > bisphenol M > BPAF>BPF > BPS > BPA. Human administered equivalent dose (AED) determination ranked BPS as the most potent alternative replacement. The benchmark concentration potency ranking of BDE-47 and organophosphate esters based on cytotoxicity was TDtBPP>BDMPP>TBOEP>TDCPP>TMPP>TPHP>BDE47>IPPP=BPDP=TCPP. Additionally, TM4 cell exposure to BDE-47 increased Calcein intensity (57.9 µM) and affected lysosomes (21.6 µM), while exposure to TPHP and TMPP resulted in cellular oxidative stress changes at benchmark concentration values as low as 0.01 and 0.4 µM, respectively. Overall bioactivity considerations of the chemicals on TM4 via ToxPi analyses and AED modeling further validated emerging replacements as highly potent chemicals in comparison to BPA and BDE-47. These findings demonstrate that many bisphenol and flame retardant replacements are more potent in Sertoli cells than the legacy chemical they are replacing and that phenotypic parameter assessment is an effective tool in chemical toxicity assessment.

Summary Sentence

In TM4 Sertoli cells, many of the chemicals that are emerging as replacements for BPA and brominated flame retardants show greater toxicity than the chemicals that they are replacing.