Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα^–/– (Esr1^fl/flWnt7a^Cre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1^fl/fl control and epiERα^–/– mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1^fl/fl and epiERα^–/– mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα^–/– uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1^fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα^–/^– vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

Summary Sentence

FOXA2 is upregulated in the uterine luminal epithelial cells and vaginal basal epithelial cells of preimplantation epiERα^–/– (Esr1^fl/flWnt7a^Cre/+) mice that are deficient of ERα in the epithelium of the female reproductive tract (FRT).

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The molecular mechanism of non-tumor female reproductive diseases is complicated and needs to be further elucidated. Recently, increasing evidence indicates that non-coding RNAs(ncRNAs) which are extremely rich in the female reproductive system are crucial factors in the pathogenesis of some female reproductive disorders. In fact, these ncRNAs such as lncRNAs, circRNAs, snoRNAs, and pseudogenes that share the same miRNA response elements (MREs) with mRNAs could compete for miRNA binding site to regulate gene expression, this phenomenon is known as the competing endogenous RNAs(ceRNAs) mechanism. This review aims to summarize the role of ceRNAs in cell proliferation, apoptosis, migration, and invasion of non-tumor female reproductive diseases such as polycystic ovary syndrome (PCOS), premature ovarian failure (POF), pre-eclampsia (PE), recurrent implantation failure (RIF), recurrent spontaneous abortion (RSA), endometriosis (EM), and endometritis, and list ceRNAs regulatory axes as well as downstream related signaling pathway. Additionally, based on certain ncRNAs that have already been proven to exist at differential levels in patient tissue samples, we also generalize some ncRNAs that can be used as potential biomarkers and therapeutic targets for these diseases in the future.

Summary Sentence

CeRNA regulatory axes and downstream signaling pathways involved in the occurrence and progression of non-tumor female reproductive diseases.

Graphical Abstract

Maternal–fetal immune tolerance is a process that involves complex interactions of the immune system, and myeloid-derived suppressor cells have emerged as one of the novel immunomodulator in the maintenance of maternal-fetal immune tolerance. Myeloid-derived suppressor cells are myeloid progenitor cells with immunosuppressive activities on both innate and adaptive cells through various mechanisms. Emerging evidence demonstrates the accumulation of myeloid-derived suppressor cells during healthy pregnancy to establish maternal–fetal immune tolerance, placentation, and fetal–growth process. By contrast, the absence or decreased myeloid-derived suppressor cells in pregnancy complications like preeclampsia, preterm birth, stillbirth, and recurrent spontaneous abortion have been reported. Here, we have summarized the origin, mechanisms, and functions of myeloid-derived suppressor cells during pregnancy along with the recent advancements in this dynamic field. We also shed light on the immunomodulatory activity of myeloid-derived suppressor cells, which can be a foundation for potential therapeutic manipulation in immunological pregnancy complications.

Summary Sentence

Myeloid-derived suppressor cells have emerged as a novel immunomodulator at the maternal–fetal interface, thus targeting myeloid-derived suppressor cells functions could positively influence the outcome of immunological pregnancy complications.

Graphical Abstract

Although assisted reproductive technology has been very successful for the treatment of infertility, its steps are still dependent on direct human opinion. An important step of assisted reproductive treatments in lab for women is choosing an oocyte that has a better quality. This step would predict which oocyte has developmental competence leading to healthy baby. Observation of the oocyte morphological quality indicators under microscope by an embryologist is the most common evaluation method of oocyte quality. Such subjective method which relies on embryologist's experience may vary and leads to misdiagnosis. An alternative solution to eliminate human misjudging in traditional methods and overcome the limitations of them is always using engineering-based procedure. In this review article, we deeply study and categorize engineering-based methods applied for the evaluation of oocyte quality. Then, the challenges in laboratories and clinics settings move forward with translational medicine perspective in mind for all those methods which had been studied were discussed. Finally, a standardized process was presented, which may help improving and focusing the research in this field. Moreover, effective suggestion techniques were introduced that are expected they would be complementary methods to accelerate future researches. The aim of this review was to create a new prospect with the engineering approaches to evaluate oocyte quality and we hope this would help infertile couples to get a baby.

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Recurrent pregnancy loss (RPL) is a common pathological problem during pregnancy, and its clinical etiology is complex and unclear. Dysfunction of trophoblasts may cause a series of pregnancy complications, including preeclampsia, fetal growth restriction, and RPL. Recently, lncRNAs have been found to be closely related to the occurrence and regulation of pregnancy-related diseases, but few studies have focused on their role in RPL. In this study, we identified a novel lncRNA BBOX1-AS1 that was significantly upregulated in villous tissues and serum of RPL patients. Functionally, BBOX1-AS1 inhibited proliferation, migration, invasion, tube formation and promoted apoptosis of trophoblast cells. Mechanistically, overexpression of BBOX1-AS1 activated the p38 and JNK MAPK signaling pathways by upregulating GADD45A expression. Further studies indicated that BBOX1-AS1 could increase the stability of GADD45A mRNA by binding hnRNPK and ultimately cause abnormal trophoblast function. Collectively, our study highlights that the BBOX1-AS1/hnRNPK/GADD45A axis plays an important role in trophoblast-induced RPL and that BBOX1-AS1 may serve as a potential target for the diagnosis of RPL.

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Obese women are subfertile and have reduced assisted reproduction success, which may be due to reduced oocyte competence. We hypothesize that consumption of a high-fat/high-sugar diet induces ovarian inflammation, which is a primary contributor to decreased oocyte quality and pre-implantation embryo development. To test this hypothesis, C57BL/6 (B6) mice with a normal inflammatory response and C3H/HeJ (C3H) mice with a dampened inflammatory response due to dysfunctional Toll-like receptor 4 were fed either normal chow or high-fat/high-sugar diet. In both B6 and C3H females, high-fat/high-sugar diet induced excessive adiposity and hyperglycemia compared to normal chow-fed counterparts. Conversely, ovarian CD68 levels and oocyte expression of oxidative stress markers were increased when collected from B6 high-fat/high-sugar but not C3H high-fat/high-sugar mice. Following in vitro fertilization of in vivo matured oocytes, blastocyst development was decreased in B6-high-fat/high-sugar but not C3H high-fat/high-sugar mice. Expression of cumulus cell markers of oocyte quality were altered in both B6 high-fat/high-sugar and C3H high-fat/high-sugar. However, there were no diet-dependent differences in spindle abnormalities in either B6 or C3H mice, suggesting potential defects in cytoplasmic maturation. Indeed, there were significant increases in the abundance of maternal effect gene mRNAs in oocytes from only B6 high-fat/high-sugar mice. These differentially expressed genes encode proteins of the subcortical maternal complex and associated with mRNA metabolism and epigenetic modifications. These genes regulate maternal mRNA degradation at oocyte maturation, mRNA clearance at the zygotic genome activation, and methylation of imprinted genes suggesting a mechanism by which inflammation induced oxidative stress impairs embryo development.

Summary Sentence

Ovarian inflammation, due to consumption of a high-fat/high-sugar diet, but not metabolic dysfunction alone, increased mRNA abundance of maternal effect genes in mature oocytes and decreased blastocyst development.

Graphical Abstract

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore–microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.

Summary Sentence

Knockdown of MARCH5 blocks mouse oocyte maturation at MI stage with the continuous activation of the spindle assembly checkpoint (SAC) and the failure of Cyclin B1 degradation, perhaps through inducing mitochondrial dysfunctions.

Graphical Abstract

Schematic diagram of MARCH5 functions in mouse oocyte maturation, as March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased ROS, decreased ATP content, and decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore–microtubule detachment. Further study showed that the continuous activation of the SAC and accumulation of MPF caused MI arrest and PB1 extrusion failure in March5 knockdown oocytes.

The formation of fertilisation-competent sperm requires spermatid morphogenesis (spermiogenesis), a poorly understood program that involves complex coordinated restructuring and specialised cytoskeletal structures. A major class of cytoskeletal regulators are the actin-related proteins (ARPs), which include conventional actin variants, and related proteins that play essential roles in complexes regulating actin dynamics, intracellular transport, and chromatin remodeling. Multiple testis-specific ARPs are well conserved among mammals, but their functional roles are unknown. One of these is actin-like 7b (Actl7b) that encodes an orphan ARP highly similar to the ubiquitously expressed beta actin (ACTB). Here we report ACTL7B is expressed in human and mouse spermatids through the elongation phase of spermatid development. In mice, ACTL7B specifically localises to the developing acrosome, within the nucleus of early spermatids, and to the flagellum connecting region. Based on this localisation pattern and high level of sequence conservation in mice, humans, and other mammals, we examined the requirement for ACTL7B in spermiogenesis by generating and characterising the reproductive phenotype of male Actl7b KO mice. KO mice were infertile, with severe and variable oligoteratozoospermia (OAT) and multiple morphological abnormalities of the flagellum (MMAF) and sperm head. These defects phenocopy human OAT and MMAF, which are leading causes of idiopathic male infertility. In conclusion, this work identifies ACTL7B as a key regulator of spermiogenesis that is required for male fertility.

Graphical Abstract

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Summary Sentence

Transcriptomic analysis from testis and epididymis of BMI by combining PacBio Iso-Seq and RNA-seq provides a comprehensive set of reference transcripts and improves the annotation of pig testis and epididymis isoforms, which will offer a valuable resource for further studying the spermatogenesis disorder in BMI caused by inbreeding.

Graphical Abstract

Ovarian follicle is the basic functional unit of female reproduction, and is composed of oocyte and surrounding granulosa cells. In mammals, folliculogenesis strictly rely on gonadotropin regulations to determine the ovulation and the quality of eggs. However, the dynamic changes of protein-expressing profiles in follicles at different developmental stages remain largely unknown. By performing mass-spectrometry-based quantitative proteomic analysis of mouse follicles, we provide a proteomic database (∼3000 proteins) that covers three key stages of gonadotropin-dependent folliculogenesis. By combining bioinformatics analysis with in situ expression validation, we showed that our proteomic data well reflected physiological changes during folliculogenesis, which provided potential to predict unknown regulators of folliculogenesis. Additionally, by using the oocyte structural protein zona pellucida protein 2 as the internal control, we showed the possibility of our database to predict the expression dynamics of oocyte-expressing proteins during folliculogenesis. Taken together, we provide a high-coverage proteomic database to study protein-expression dynamics during gonadotropin-dependent folliculogenesis in mammals.

Summary Sentence

Mass-spectrometry-based quantitative proteomic analysis provides a database of the protein-expression profiles during gonadotropin-dependent folliculogenesis in mice.

Graphical Abstract

Lipid droplets (LDs) are endoplasmic reticulum (ER)-derived organelles comprising a core of neutral lipids surrounded by a phospholipid monolayer. Lipid droplets play important roles in lipid metabolism and energy homeostasis. Mammalian ovaries have been hypothesized to use neutral lipids stored in LDs to produce the hormones and nutrients necessary for rapid follicular development; however, our understanding of LD synthesis remains incomplete. In this study, we generated transgenic reporter mice that express mCherry fused to HPos, a minimal peptide that localizes specifically to nascent LDs synthesized at the ER. With this tool for visualizing initial LD synthesis in ovaries, we found that LDs are synthesized continuously in theca cells but rarely in inner granulosa cells (Gc) during early follicular development. Administration of exogenous gonadotropin enhances LD synthesis in the Gc, suggesting that LD synthesis is hormonally regulated. In contrast, we observed copious LD synthesis in the corpus luteum, and excessive LDs accumulation in atretic follicles. Furthermore, we demonstrated that LD synthesis is synchronized with angiogenesis around the follicle and that suppressing angiogenesis caused defective LD biosynthesis in developing follicles. Overall, our study is the first to demonstrate a spatiotemporally regulated interplay between LD synthesis and neovascularization during mammalian follicular development.

Summary Sentence

Analysis of ovaries from transgenic reporter mice expressing mCherry fused to HPos, which localizes specifically to nascent lipid droplets (LDs), revealed that spatiotemporally regulated LD synthesis is closely associated with angiogenesis.

Graphical Abstract

The cause for at least 50% of recurrent miscarriages is unclear, which is defined as unexplained recurrent miscarriages. The B7-H1 (PD-L1), a molecule of the B7 family, promotes tumor development by modulating immune evasion, and recent researchers have also attached importance to the role of B7-H3, another molecule of B7 family, in tumor. Based on the similarity between growth and immune response in tumors and pregnancy, we first explored the role of B7-H3 in unexplained recurrent miscarriages. We found reduced levels of B7-H3 in the villus tissue of unexplained recurrent miscarriage patients, and it was mainly expressed on the cell membrane of extravillous trophoblasts. Further, the HTR-8/SVneo and JEG-3 cells were selected to explore the role of B7-H3 in proliferation, apoptosis, tube formation, migration, and invasion. We found that B7-H3 regulated trophoblast migration and invasion via RhoA/ROCK2 signaling pathway. Inflammatory cytokines were detected through enzyme-linked immunosorbent assay after co-culturing with decidual natural killer cells and B7-H3-knockout JEG-3. Results showed that B7-H3 inhibited IL-8 and IP-10 secretion from the decidual natural killer cells. In a CBA/J × DBA/2 abortion-prone mice model, treatment with B7-H3-Fc protein successfully reduced the rate of embryo resorption. In conclusion, our results revealed a possible mechanism by which decreased B7-H3 on trophoblasts of unexplained recurrent miscarriages inhibited trophoblast migration and invasion and increased IL-8 and IP-10 secretion from the decidual natural killer cells. Furthermore, B7-H3 may be a promising new therapeutic target in unexplained recurrent miscarriage patients.

Summary Sentence

Decreased B7-H3 participates in unexplained recurrent miscarriage by inhibiting trophoblast migration and invasion via RhoA/ROCK2 pathway and regulating decidual NK secretion.

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