With the development of the embryo, the totipotent blastomere undergoes the first lineage decision to the inner cell mass (ICM) and the trophectoderm (TE). The ICM forms the fetus while the TE forms the placenta, which is one of the unique organs in mammals serving as the interface between maternal and fetal bloodstreams. Proper trophoblast lineage differentiation is crucial for correct placental and fetal development, including the TE progenitor self-renewal and its differentiation toward mononuclear cytotrophoblast, which later either develops into invasive extravillous trophoblast, remodeling the uterine vascular, or fuses into multinuclear syncytiotrophoblast, secreting pregnancy-sustaining hormone. Aberrant differentiation and gene expression of trophoblast lineage is associated with severe pregnancy disorders and fetal growth restriction. This review focuses on the early differentiation and key regulatory factors of trophoblast lineage, which have been poorly elucidated. Meanwhile, the recent development of trophoblast stem cells, trophectoderm stem cells, and blastoids derived from pluripotent stem cells bring the accessible model to investigate the profound mystery of embryo implantation and placentation and were also summarized.

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Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide–dependent deacetylase that modifies gene expression through histone deacetylation. It also deacetylates nonhistone substrates, e.g., tumor suppressor p53, NOS3, HIF1A, NFKB, FOXO3a, PGC-1α, and PPARγ . Consequently, it regulates a wide range of physiological functions including cell cycle control, energy expenditure, oxidative stress response, apoptosis, and aging. SIRT1 is expressed in ovarian granulosa cells (GCs) of various species including humans at different stages of the reproductive cycle. The importance of SIRT1 in female reproduction is supported by the findings that SIRT1-knockout mice exhibit defects in reproductive tissue development. These mice were found to have a thin-walled uterus, small ovaries, with follicles present but no corpora lutea. This review aims to provide state-of-the-art information on SIRT1's mode of action and its roles in human granulosa-lutein cells and GCs from other species where data are available. It also discusses the overlapping actions of SIRT1 and human chorionic gonadotropin on the production of critical GC-borne factors.

Summary Sentence

SIRT1 regulates the expression of a battery of genes known to affect angiogenesis, ovulation, and glucose metabolism in human luteinized GCs as well as their survival, most likely in a cAMP-dependent manner.

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Illustrative summary depicting the diverse actions of SIRT1 in luteinized human granulosa cells. SIRT1, activated by SRT2104, was previously shown to elevate cAMP levels [21]. Therefore, SIRT1-induced genes are pivotal for ovulation (EREG and PTGS2), angiogenesis (FGF2 and VEGFA), and glucose metabolism (SLC2A1), most likely in a cAMP-dependent manner. SIRT1 activation and the subsequent elevation of cAMP may also promote the reduction of EDN2 and anti-apoptotic proteins BCL-XL and MCL1. This triggers the cleavage and activation of caspase 3. Cleaved caspase 3 then cleaves PARP, consequently leading to apoptosis. One may therefore suggest that SIRT1-induced cAMP is responsible for the coexistence of luteinization and apoptosis in luteinizing GCs. SIRT1 also activates RIPK1 and MLKL proteins, thereby advancing necroptosis. In addition, activation of RIPK1 can also mediate apoptosis by activating caspase 3. The role of RIPK1 in SIRT1-induced apoptosis and necroptosis is manifested by the attenuation of apoptotic and necroptotic proteins by Nec-1. The patterns of FACS-sorted cells further support the role of SIRT1 as an inducer of apoptosis and necroptosis. Ablation of endogenous SIRT1 with siRNA produces opposing actions, thereby corroborating the effects observed with SIRT1 activation. Adapted from [51].

To understand the effect of fetal thyroid gland disruption on development in swine, we evaluated thyroid hormone levels, growth and developmental characteristics, and gene expression associated with thyroid hormone metabolism in late gestation fetuses exposed to methimazole (MMI). Pregnant gilts were given either oral MMI or equivalent sham from gestation day 85–106 (n = 4/group), followed by intensive phenotyping of all fetuses (n = 120). Samples of liver (LVR), kidney (KID), fetal placenta (PLC), and the corresponding maternal endometrium (END) were collected from a subset of fetuses (n = 32). Fetuses exposed to MMI in utero were confirmed hypothyroid, with a significant increase in thyroid gland size, goitrous thyroid histology, and dramatically suppressed thyroid hormone in serum. In dams, no differences in temporal measurements of average daily gain, thyroid hormone, or rectal temperatures relative to controls suggests that MMI had little effect on maternal physiology. However, fetuses from MMI-treated gilts exhibited significant increases in body mass, girth, and vital organ weights, but no differences in crown-rump length or bone measurements suggesting non-allometric growth. The PLC and END showed a compensatory decrease in expression of inactivating deiodinase (DIO3). Similar compensatory gene expression was observed in fetal KID and LVR with a downregulation of all deiodinases (DIO1, DIO2, DIO3). Minor alterations in the expression of thyroid hormone transporters (SLC16A2 and SLC16A10) were observed in PLC, KID, and LVR. Collectively, MMI crosses the PLC of the late gestation pig, resulting in congenital hypothyroidism, alterations in fetal growth, and compensatory responses within the maternal fetal interface.

Summary Sentence

Methimazole crosses the late gestation pig placenta resulting in congenital hypothyroidism, non-allometric fetal growth, and compensatory changes in thyroid hormone metabolites.

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To investigate the expression profiles of circular RNAs (circRNAs) in the endometria of women with polycystic ovary syndrome (PCOS) and to explore the role of aberrant circ_0115118 expression in endometrial dysfunction in patients with PCOS. CircRNA microarray hybridization and bioinformatic analyses were performed to determine the expression patterns of circRNAs in the endometria of patients with or without PCOS, the expression of target circRNA was evaluated by real-time polymerase chain reaction (PCR). Cell counting kit-8 and Transwell assays were used to detect cellular proliferative, invasive, and migratory capacities. The influence of the circRNA on decidualization was explored by real-time PCR. Animal models were established to investigate the regulatory effect of the circRNA on embryo implantation. Downstream microRNAs and genes were predicted using bioinformatic websites and verified by dual-luciferase reporter assays, real-time PCR, and western blotting. In the endometria of patients with PCOS, there were 113 differentially expressed circRNAs in the secretory phase and 1119 differentially expressed circRNAs in the proliferative phase. The expression of circ_0115118 was significantly higher in endometrial stromal cells during the proliferative phase in patients with PCOS, leading to inhibition of cellular mobilization and embryo implantation. In addition, circ_0115118 exerted effects by sponging miR-138-1-3p, subsequently increasing the expression of WD repeat and FYVE domain-containing protein 2 (WDFY2). Circ_0115118 expression is dysregulated in the endometria of patients with PCOS and adversely affects endometrial function. Our findings reveal that circ_0115118 may be a potential therapeutic target to improve pregnancy outcomes in women with PCOS.

Summary Sentence

Aberrantly upregulated expression of circ_0115118 in endometria of PCOS patients hindered normal cellular activities by targeting the miR-138-1-3p/WDFY2 axis, thus influencing embryo implantation.

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Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFβ were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220⁺) B cells and mature (CD138⁺) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Summary Sentence

Pro-inflammatory and regulatory T cells and B cells secreting IgG and IgA are elicited by prophylactic and therapeutic vaccination to reduce male reproductive tract chlamydial burden, protecting the testicular environment and spermatogenesis from damage normally induced by chlamydial infection.

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P450 aromatase, encoded by the Cyp19 gene, catalyzes the synthesis of estrogen, which is crucial for mammalian germ cell differentiation. We have previously shown that transforming growth factor beta 1 (TGF-β1) attenuated the accumulation of steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) and eventually reduced the transcription of Cyp19 in rat Leydig cells (LCs). Here, we report that TGF-β1 treatment-induced phosphorylation of Smad2 and decreased the expression levels of SF-1 and LRH-1 by elevating the expression levels of microRNA-21-3p and microRNA-339-5p in vivo and in vitro. Furthermore, both TGF-β1 treatment and over-expression of Smad2 inhibited the SF-1 or LRH-1-regulated promoter activity of the Cyp19 gene, and p-Smad2 physically interacted with SF-1 and LRH-1. Our findings collectively suggest that TGF-β1 may inhibit the expression of CYP19 in LCs mainly through two ways. On the one hand, TGF-β1 acts through Smad2 to repress the accumulation of SF-1 and LRH-1 at post-transcriptional level by upregulating specific microRNAs. On the other hand, TGF-β1 inhibits the transcriptional activity of Cyp19 through the interaction of p-Smad2 with SF-1/LRH-1.

Summary Sentence

TGF-β1 inhibits Cyp19 transcription mediated by Smad2 in two ways: upregulation of miRNAs targeting SF-1/LRH-1 and inactivation of Cyp19 promoter.

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Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis (transcriptome_analysis_ioad014.xls). Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-KB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-KB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIKBα/IKBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIKBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-KB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

Summary Sentence

Endometriosis showed repression of NF-KB pathway and downregulation of HPGD and COX2, which may be involved in ovulation by participating in the metabolism of prostaglandin E2 (PGE2), in GCs due to elevated progesterone in FF.

Some transmasculine individuals may be interested in pausing gender-affirming testosterone therapy and carrying a pregnancy. The ovarian impact of taking and pausing testosterone is not completely understood. The objective of this study was to utilize a mouse model mimicking transmasculine testosterone therapy to characterize the ovarian dynamics following testosterone cessation. We injected postpubertal 9–10-week-old female C57BL/6N mice once weekly with 0.9 mg of testosterone enanthate or a vehicle control for 6 weeks. All testosterone-treated mice stopped cycling and demonstrated persistent diestrus within 1 week of starting testosterone, while control mice cycled regularly. After 6 weeks of testosterone therapy, one group of testosterone-treated mice and age-matched vehicle-treated diestrus controls were sacrificed. Another group of testosterone-treated mice were maintained after stopping testosterone therapy and were sacrificed in diestrus four cycles after the resumption of cyclicity along with age-matched vehicle-treated controls. Ovarian histological analysis revealed stromal changes with clusters of large round cells in the post testosterone group as compared to both age-matched controls and mice at 6 weeks on testosterone. These clusters exhibited periodic acid–Schiff staining, which has been previously reported in multinucleated macrophages in aging mouse ovaries. Notably, many of these cells also demonstrated positive staining for macrophage markers CD68 and CD11b. Ovarian ribonucleic acid-sequencing found upregulation of immune pathways post testosterone as compared to age-matched controls and ovaries at 6 weeks on testosterone. Although functional significance remains unknown, further attention to the ovarian stroma may be relevant for transmasculine people interested in pausing testosterone to carry a pregnancy.

Summary Sentence

After testosterone is paused for reproductive purposes in a transgender mouse model, estrous cyclicity resumes, but ovaries demonstrate stromal aberrations and an upregulated inflammatory reaction.

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Women are born with hundreds of thousands to over a million primordial ovarian follicles (PFs) in their ovarian reserve. However, only a few hundred PFs will ever ovulate and produce a mature egg. Why are hundreds of thousands of PFs endowed around the time of birth when far fewer follicles are required for ongoing ovarian endocrine function and only a few hundred will survive to ovulate? Recent experimental, bioinformatics, and mathematical analyses support the hypothesis that PF growth activation (PFGA) is inherently stochastic. In this paper, we propose that the oversupply of PFs at birth enables a simple stochastic PFGA mechanism to yield a steady supply of growing follicles that lasts for several decades. Assuming stochastic PFGA, we apply extreme value theory to histological PF count data to show that the supply of growing follicles is remarkably robust to a variety of perturbations and that the timing of ovarian function cessation (age of natural menopause) is surprisingly tightly controlled. Though stochasticity is often viewed as an obstacle in physiology and PF oversupply has been called “wasteful,” this analysis suggests that stochastic PFGA and PF oversupply function together to ensure robust and reliable female reproductive aging.

Summary Sentence

Mathematical extreme value theory analysis of human primordial follicle numbers over time supports the hypothesis that the large endowment of primordial follicles at birth enables a simple stochastic PFGA mechanism to yield a steady supply of growing follicles that lasts for several decades.

The foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia, which comprise of spermatogonial stem cells (SSCs) and transit-amplifying progenitors that differentiate in response to retinoic acid (RA) and are committed to enter meiosis. Our laboratory recently reported that the foundational populations of SSCs, undifferentiated progenitors, and differentiating spermatogonia are formed in the neonatal testis in part based on their differential responsiveness to RA. Here, we expand on those findings to define the extent to which RA responsiveness during steady-state spermatogenesis in the adult testis regulates the spermatogonial fate. Our results reveal that both progenitor and differentiating spermatogonia throughout the testis are capable of responding to exogenous RA, but their resulting fates were quite distinct—undifferentiated progenitors precociously differentiated and proceeded into meiosis on a normal timeline, while differentiating spermatogonia were unable to hasten their entry into meiosis. This reveals that the spermatogonia responding to RA must still complete the 8.6 day differentiation program prior to their entry into meiosis. Addition of exogenous RA enriched testes with preleptotene and pachytene spermatocytes one and two seminiferous cycles later, respectively, supporting recent clinical studies reporting increased sperm production and enhanced fertility in subfertile men on long-term RA analog treatment. Collectively, our results reveal that a well-buffered system exists within mammalian testes to regulate spermatogonial RA exposure, that exposed undifferentiated progenitors can precociously differentiate, but must complete a normal-length differentiation program prior to entering meiosis, and that daily RA treatments increased the numbers of advanced germ cells by directing undifferentiated progenitors to continuously differentiate.

Summary Sentence

Retinoic acid induces widespread precocity of progenitors, not differentiating spermatogonia, through steady-state spermatogenesis

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The use of bis (2-ethylhexyl) phthalate (DEHP), 2,2′4,4′-tetrabromodiphenyl ether (BDE47), and bisphenol A (BPA), as plasticizers, flame retardants, and epoxy resins, respectively, has been regulated due to their endocrine disrupting activities. Replacements for these chemicals are found in human matrices, yet the endocrine disrupting potential of these emerging contaminants is poorly characterized. We compared the effects of legacy chemicals with those of their replacements using fetal rat testis organ culture. Fetal testes sampled at gestation day 15 were grown ex vivo, and the impact was evaluated after a 3-day exposure to 10 µM of each legacy chemical; two BPA analogs (bisphenol M and bisphenol TMC); three replacements for DEHP/MEHP (2,2,4-trimethyl-1,3-pentanediol diisobutyrate, diisononyl-phthalate, and diisodecyl adipate); or two replacements for BDE47 (tributoxyethyl phosphate and isopropylated triphenyl phosphate). We showed that only BPA and MEHP significantly decrease testosterone secretions after 24 h, while BPM and BPTMC have the opposite effect. Luteinizing hormone-stimulated testosterone was reduced by BPA and MEHP but was increased by BPTMC. After exposure, testes were used for immunofluorescent staining of germ cells, Sertoli cells, and Leydig cells. Interestingly, exposures to BPM or BPTMC induced a significant increase in the Leydig cell density and surface area. A decrease in germ cell density was observed only after treatment with MEHP or BDE47. MEHP also significantly decreased Sertoli cell proliferation. These studies show that some replacement chemicals can affect testicular function, while others appear to show little toxicity in this model. These findings provide essential information regarding the need for their regulation.

Summary Sentence

In the fetal rat testis, while bis (2-ethylhexyl) phthalate and 2,2′4,4′-tetrabromodiphenyl ether replacements appear less toxic, emerging bisphenol A substitutes stimulate steroidogenesis and affect fetal Leydig cell physiology.

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