The first anti-Müllerian hormone (AMH) radioimmunoassay became available for clinical use in the early 2000s. This MHR Legacy Series review discusses key contributions from our own group towards establishing AMH as a robust ovarian reserve biomarker. Initial studies in female volunteers demonstrated decreasing serum AMH concentrations with increasing female age, along with a direct correlation between AMH levels and the number of antral follicles. The most relevant clinical conditions where measurements of serum AMH appear to be of great value include the diagnosis of various forms of ovarian dysfunction, especially polycystic ovary syndrome and premature ovarian insufficiency. In addition, a close correlation between initial serum AMH levels and ovarian response to standard stimulation for IVF has been firmly established, resulting in the development of a safe and effective AMH-based algorithm for individualized ovarian stimulation. Another area, beyond our own research, where AMH serum measurements have proven to be of great clinical significance is oncofertility. The capacity of AMH levels to predict fecundity-either spontaneous or following infertility treatment-or forecast age of menopause later in life remains uncertain and needs more well-designed, prospective, follow-up studies.

Adenomyosis (AM) is traditionally explained by biochemical dysregulation at the endometrial-myometrial interface, leading to passive displacement of endometrial stromal cells (ESCs). However, the uterus is a highly mechanosensitive organ. Aberrant myometrial hypercontractility in AM may expose ESCs to chronic mechanical stimulation. Whether such mechanical cues can modulate ESCs' behavior under in vitro conditions remains incompletely understood. This study hypothesizes that cyclic mechanical strain-independent of biochemical cues-can induce a pro-migratory and pro-invasive ESC phenotype in vitro. Immortalized human ESCs (hESCs) were subjected to cyclic uniaxial stretching (12% strain, 0.2 Hz, 48 h) to model sustained mechanical loading. We evaluated cellular morphology, migration and invasion (wound healing and Transwell assays), cytoskeletal remodeling (F-actin), extracellular matrix (ECM) related proteins, transcriptomic changes (RNA-sequencing), and MAPK pathway activation. The functional involvement of p38 MAPK was tested using the selective inhibitor Adezmapimod. The results indicated that cyclic stretch induced marked cellular elongation without increasing apoptosis. RNA-sequencing revealed 580 differentially expressed genes enriched in migration and ECM remodeling. Functionally, stretched hESCs exhibited enhanced migration and invasion capacity in vitro, accompanied by elevated F-actin, fibronectin, and CTGF. Mechanistically, mechanical strain triggered selective activation of the p38 MAPK pathway, without affecting ERK or JNK. Pharmacological inhibition of p38 attenuated stretch-associated cytoskeletal remodeling, ECM-related protein expression, and cell motility. Together, these findings demonstrate that cyclic mechanical stretch, as an underexplored non-biochemical factor, can promote a motile and invasive stromal cell phenotype in vitro via a p38 MAPK/F-actin-associated mechanism. While these results do not establish causality for AM development in vivo, they suggest a potential mechanotransduction pathway through which abnormal uterine mechanics may influence stromal cell behavior. Targeting mechanotransduction-such as p38 inhibition-may represent novel mechano-therapeutics for AM. This warrants further investigation using more complex and physiologically relevant models.

Male infertility remains unexplained in a substantial proportion of cases, despite extensive clinical and biological investigations. Oxidative stress has emerged as a central pathophysiological mechanism underlying impaired sperm function, yet current diagnostic and therapeutic strategies inadequately capture the complexity of redox regulation within the male reproductive tract. In this narrative review, we revisit both the historical foundations and recent advances supporting a pivotal role for a specific class of metabolites: polyamines (spermine, spermidine, and putrescine) and carnitine, which we propose to collectively term antioxidant amines. These compounds are present at exceptionally high concentrations in seminal plasma, tightly regulated by androgen-dependent and metabolic pathways, and critically involved in redox homeostasis, mitochondrial function, chromatin stabilization, and sperm maturation. We synthesize evidence from classical biochemical studies, contemporary metabolomics, and translational research linking antioxidant amine imbalance to altered semen parameters and male subfertility. Importantly, we highlight recent analytical advances that now make the targeted quantification of these metabolites technically feasible in clinical laboratories using routinely available liquid chromatography-mass spectrometry (LC-MS) platforms. Beyond their diagnostic potential, we critically discuss experimental and clinical data on antioxidant amine supplementation, emphasizing both their therapeutic potential and the need for balanced dosing strategies to avoid deleterious effects associated with excess exposure. Altogether, this review positions antioxidant amines at the crossroads of pathophysiology, laboratory medicine, and clinical intervention in male infertility. By integrating historical insights with modern analytical and translational perspectives, we argue that antioxidant amines are finally ready to move from overlooked metabolites to actionable biomarkers and therapeutic targets in reproductive medicine.

Fertilization is the process by which sperm and oocyte recognize each other and fuse to form a zygote. Fertilization failure is a common issue encountered in ART. However, clinically, there is a lack of identification of the specific stage at which fertilization is arrested and the development of personalized improvement strategies. In this study, we conducted a fertilization failure assay (FFA) in a cohort of 445 couples undergoing IVF (n = 394) and ICSI (n = 51) who experienced total fertilization failure (TFF). In subsequent cycles, comparative analysis was performed to assess both fertilization rates and good-quality embryo rates between couples at different arrest stages undergoing either conventional ICSI or ICSI with assisted oocyte activation (ICSI-AOA). Additionally, whole-exome sequencing (WES, n = 20) was implemented to investigate potential genetic mutations associated with specific fertilization arrest stages. It found that unfertilized oocytes were categorized into four fertilization arrest phenotypes: (i) sperm penetration/fusion deficiency (SPD), (ii) histone incorporation deficiency (HID), (iii) pronuclear-formation deficiency (PFD), and (iv) mixed deficiency. SPD was the primary cause of TFF after IVF (67.8%), and this deficiency was circumvented in ICSI cycles. HID and PFD were the causes of TFF after ICSI. Couples with ≥50.0% oocytes arrested at the histone incorporation stage or ≥75.7% oocytes arrested at the pronuclear formation stage were at high risk of low fertilization rate following ICSI. Couples with ≥50.0% of oocytes arrested at the histone incorporation stage may achieve improved fertilization and embryo outcomes through ICSI-AOA. Genetic mutations associated with male-factor etiologies of fertilization failure have been identified in this patient population. In contrast, for couples with arrest at the PFD, ICSI-AOA shows no beneficial effects on fertilization or embryo development. Genetic mutations associated with female-factor etiologies of fertilization failure have been identified in this patient population. In conclusion, FFA delineates the arrest stages during fertilization failure, facilitates analysis of insemination progression, and provides clinical reference for subsequent treatment cycles.

Mammalian oocyte maturation is a tightly regulated process essential for successful fertilization and embryonic development. Meiotic resumption in mammalian oocytes is controlled by various regulatory factors, including the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHA/14-3-3). However, the specific functions of individual YWHA isoforms in oocyte meiosis remain poorly understood. In this study, we revealed that knockdown of Ywhaz, one of the isoforms of YWHA, using short interfering RNA (siRNA) or morpholino oligomers (MOs), accelerates meiotic resumption in mouse oocytes. To elucidate the mechanism underlying YWHAZ-mediated meiotic resumption, we thus explored its interactions with potential target proteins. Co-immunoprecipitation and immunofluorescence analyses demonstrated a physical interaction between YWHAZ and phosphorylated CDC25B. Additionally, we identified the protein kinases responsible for YWHAZ phosphorylation at distinct residues. Specifically, JNK1, CSNK1A1/CKIα, and protein kinase B (PKB/Akt) were found to phosphorylate YWHAZ at Serine 184/186, Threonine 232, and Serine 58, respectively. Notably, phosphorylation of YWHAZ at serine 58 by PKB/Akt promoted meiotic resumption in mouse oocytes. Furthermore, we found the formation of a heterodimer between YWHAZ and YWHAQ. Our results provide insights into the PKB/Akt-YWHAZ-CDC25B signalling pathway and illuminate the functional influence of YWHAZ phosphorylation in meiotic regulation.

We have previously shown that ligustilide (LIG) treatment can restore endometrial receptivity and promote implantation in pregnant rats with low endometrial receptivity (LER). Using mRNA sequencing technology, we found that desmoglein-2 (Dsg2) is one of the differentially expressed genes, which is downregulated in rats with LER but elevated after LIG treatment. DSG2 is an adhesion protein that exists in epithelial tissues and serves as a crucial component of the junction complex for intercellular adhesion. The ability to adhere between cells is considered essential for successful embryo implantation. Hence, we speculated that DSG2 might be involved in the mechanism by which LIG treats LER. Immunofluorescence staining, western blots, and immunohistochemical staining were performed to confirm the restored expression of DSG2 by LIG treatment in the endometrial tissues of LER rats. Then, we investigated the effect of DSG2 on endometrial receptivity by injecting small interfering (si)Dsg2 (for silencing Dsg2 expression) into one uterine horn of a pregnant rat and siNC (for a normal control) into the other. It was found that the side of the endometrium injected with siDsg2 showed significant changes in the expression of multiple receptivity-related molecules and obvious embryo loss. In addition, we found that these receptivity-related molecules were affected by siDsg2 in Ishikawa (an endometrial cell line) cells. Dsg2 knockdown reduced the ability of Ishikawa cells to adhere to trophoblastic spheroids. Furthermore, the effect of LIG on endometrial cell-trophoblast adhesion was partially abolished by Dsg2 silencing. In conclusion, our findings suggest that the protective effect of LIG against LER might be achieved by increasing DSG2 expression. DSG2 enhancement might become a novel strategy for the development of LIG-inspired treatments to improve endometrial receptivity.

Maternal diet-induced obesity (DIO) may affect adult offspring oocyte quality due to mitochondrial dysfunction. Here, we investigated whether offspring of DIO mothers exhibit mitochondrial abnormalities in their primordial follicle oocytes (PFOs) already at birth and if (further) alterations can be detected at weaning. Female Swiss mice were fed a control or obesogenic diet for 7 weeks before mating and throughout pregnancy and lactation. Offspring ovaries were collected at birth and at weaning. Offspring PFOs were examined by transmission electron microscopy of ovarian sections. Key markers of cell stress (HSP70), mitochondrial biogenesis (PGC-1α), mtDNA replication (TFAM), fusion (MFN2, OPA1), and fission (DRP1) were examined using immunofluorescence and confocal microscopy. Maternal DIO did not alter HSP70 or PGC-1α expression in the PFOs at birth, suggesting that cellular homeostasis and mitochondrial biogenesis were unaffected. TFAM expression was reduced at both time points. DRP1 and cytoplasmic OPA1 expression were reduced at birth, but without ultrastructural changes in mitochondrial shape and density, suggesting that these alterations are regulatory. No inborn mitochondrial structural abnormalities could be detected. In contrast, at weaning, offspring born to and nursed by DIO mothers exhibited a high number of lipid droplets (LDs) in their ovaries, some of which were detectable in the PFOs, while no LDs were detected in the PFOs of the controls. Maternal DIO increased PGC-1α expression, suggesting postnatal effects on PFO mitochondrial biogenesis. MFN2 and OPA1 expression also increased, together with increased mitochondrial elongation and a reduced mitochondrial density. Mitochondrial abnormalities, such as vacuolation, loose inner membranes, the number of detected autophagosomes, and signs of lipophagy, were also significantly increased by maternal DIO at weaning. In conclusion, the oocyte mitochondrial structural abnormalities previously reported in adult offspring from DIO mothers were not detected in the PFOs at birth. Significant changes in primordial follicles linked to maternal DIO were detected only at weaning.

Katanins are microtubule severing enzymes that play roles in diverse cell functions including meiotic and mitotic spindle formation. To address the role of Katanin p60 isozymes in mammalian oocytes, we have used the ZP3-CreLox approach to specifically delete Katanin A1 (KATNA1) and Katanin A-like 1 (KATNAL1) from the start of oocyte growth. Here, we show that KATNAL1 is not required for normal fertility, but deletion of KATNA1 causes a 50% decrease in fertility. Further investigation in Katna1−/− oocytes revealed no effect on MI spindle morphology but a modest effect on the morphology of MII spindles. This was accompanied by a decreased rate of fertilization, but Katna1−/+ heterozygous embryos that reached the 2-cell stage developed at normal rates to the blastocyst stage. Parthenogenetic activation of Katna1−/− oocytes to generate diploid homozygous embryos revealed a reduced rate of blastocyst formation. Further, the Katna1−/− parthenogenetic blastocysts had a reduced diameter, decreased cell number, and increased nuclear size. Taken together, our data indicate KATNA1, but not KATNAL1, plays a role in MII spindle function and mitotic cell divisions of the preimplantation embryo. The ability of the paternal allele to rescue preimplantation development suggests the origin of the decrease in the fertility of conditional Katna1−/− mice lies in abnormalities arising in the egg to embryo transition prior to embryonic genome activation.

Leiomyomas are benign proliferations of uterine smooth muscle found in 60% of women. A spatial redistribution of ecto-5'-nucleotidase (CD73, NT5E) that results in reduced extracellular concentrations of adenosine has recently been described in leiomyomas. However, the mechanisms by which altered extracellular adenosine levels contribute to leiomyoma growth remain poorly understood. To address this deficiency, a series of tissue specimens and primary cultures generated from matched specimens of myometrium and leiomyoma were used. Overexpression of Type 1 adenosine receptors (ADORA1) was observed when matched specimens and primary cultures were interrogated by RT-qPCR and western blot. By immunohistochemistry, ADORA1 expression was diffusely observed in myocytes in the leiomyoma complex, with only limited expression in vascular and other structures. Overexpression of ADORA1 was also observed in fibroblasts and multiple smooth muscle subtypes in the leiomyoma complex when single-cell transcriptomics data were interrogated. Incubation with N6-cyclopentyladenosine (CPA), a selective ADORA1 agonist, resulted in decreased proliferation of primary leiomyoma cultures, accompanied by decreased intracellular cAMP and enhanced cyclin D1 and phospho-AKT1 expression. To confirm the specificity of this observation, ADORA1 expression was directly targeted by siRNA, resulting in decreased proliferation, increased intracellular cAMP, and lower levels of cyclin D1 and phospho-AKT1. Collectively, these data indicate that overexpression of the ADORA1 receptor is a robust feature of uterine leiomyomas, where its activation by residual levels of extracellular adenosine potentially contributes to tumor growth by regulating AKT1-mediated signaling.