Organ functions generally decline with age, but the ovary is a prototypical organ that undergoes functional loss over time. Autophagy plays a crucial role in maintaining organ homeostasis, and age-related upregulation of the autophagy inhibitor protein, Rubicon, has been linked to cellular and tissue dysfunction. This review describes how granulosa cell autophagy supports follicular growth and oocyte selection and maturation by regulating cellular energy metabolism and protein quality control. We then introduce the role of selective autophagy, including mitophagy or lipophagy, in steroidogenesis and cellular remodeling during luteinization. In aged ovaries, Rubicon accumulation suppresses autophagic flux, leading to diminished oxidative-stress resilience and enhanced DNA damage. Moreover, impaired autophagy drives the accumulation of ATP citrate lyase, which correlates with poor oocyte quality and reduced ovarian reserve. Following fertilization, oocytes further upregulate autophagy to provide the energy required for blastocyst transition. Conversely, in infertility-related disorders, such as premature ovarian insufficiency, endometriosis, and polycystic ovary syndrome, either deficient or excessive autophagy contributes to disease pathogenesis. Both autophagy inhibitors (e.g., Rubicon) and activators (e.g., Beclin1) could be emerging as promising biomarkers for assessing ovarian autophagy status. Therapeutically, Rubicon inhibition by trehalose in aged ovaries and autophagy suppression by agents such as hydroxychloroquine in polycystic ovary syndrome and endometriosis hold potential. Establishing robust methods to evaluate ovarian autophagy will be essential for translating these insights into targeted treatments.

Anti-β2-glycoprotein I/HLA-DR (anti-β2GPI/HLA-DR) antibody has been reported to be associated with antiphospholipid syndrome and recurrent pregnancy loss (RPL). We conducted a prospective multicenter cross-sectional study aimed at evaluating whether the anti-β2GPI/HLA-DR antibody is associated with adverse obstetric outcomes and RPL. From 2019 to 2021, serum anti-β2GPI/HLA-DR antibody levels (normal, <73.3 U) were measured in 462 women with RPL, 124 with fetal growth restriction (FGR), 138 with hypertensive disorders of pregnancy (HDP), 71 with preterm delivery before 34 gestational weeks (preterm delivery (PD) ≤ 34 GWs), and 488 control women who experienced normal delivery, by flow cytometry analysis. The adjusted odds ratios (aORs) of anti-β2GPI/HLA-DR antibody positivity for adverse obstetric outcomes and RPL were evaluated on the basis of comparisons between the control and each patient group, using multivariable logistic regression analysis. The following were the positivity rates for the anti-β2GPI/HLA-DR antibody in the patient and control groups: RPL, 16.9%; FGR, 15.3%; HDP, 17.4%; PD ≤ 34 GWs, 11.3%; and the control, 5.5%. It was demonstrated that anti-β2GPI/HLA-DR antibody positivity was a significant risk factor for RPL (aOR, 3.3 [95% confidence interval {CI} 1.9–5.6], p < 0.001), FGR (2.7 [1.3–5.3], p < 0.01), and HDP (2.7 [1.4–5.3], p < 0.01) although not for PD ≤ 34 GWs. For the first time, our study demonstrated that the anti-β2GPI/HLA-DR antibody is involved in the pathophysiology underlying FGR and HDP, as well as RPL.

Organ functions generally decline with age, but the ovary is a prototypical organ that undergoes functional loss over time. Autophagy plays a crucial role in maintaining organ homeostasis, and age-related upregulation of the autophagy inhibitor protein, Rubicon, has been linked to cellular and tissue dysfunction. This review describes how granulosa cell autophagy supports follicular growth and oocyte selection and maturation by regulating cellular energy metabolism and protein quality control. We then introduce the role of selective autophagy, including mitophagy or lipophagy, in steroidogenesis and cellular remodeling during luteinization. In aged ovaries, Rubicon accumulation suppresses autophagic flux, leading to diminished oxidative-stress resilience and enhanced DNA damage. Moreover, impaired autophagy drives the accumulation of ATP citrate lyase, which correlates with poor oocyte quality and reduced ovarian reserve. Following fertilization, oocytes further upregulate autophagy to provide the energy required for blastocyst transition. Conversely, in infertility-related disorders, such as premature ovarian insufficiency, endometriosis, and polycystic ovary syndrome, either deficient or excessive autophagy contributes to disease pathogenesis. Both autophagy inhibitors (e.g., Rubicon) and activators (e.g., Beclin1) could be emerging as promising biomarkers for assessing ovarian autophagy status. Therapeutically, Rubicon inhibition by trehalose in aged ovaries and autophagy suppression by agents such as hydroxychloroquine in polycystic ovary syndrome and endometriosis hold potential. Establishing robust methods to evaluate ovarian autophagy will be essential for translating these insights into targeted treatments.

A revised two-stage model of preeclampsia is proposed, centering on an autophagy-dependent requirement for extravillous trophoblast entry into the proximal one-third of the myometrium. The One-Third Myometrium Enigma, introduced here, denotes the unresolved physiological rule that early placentation requires trophoblasts to traverse decidua and reach the proximal one-third of myometrium under hypoxia and nutrient scarcity. The hypothesis posits a timed rise in basal autophagy to sustain trophoblast energy homeostasis and invasion, accompanied by TFEB-driven lysosomal programs that enable villous cytotrophoblast syncytialization. Autophagic dysfunction could contribute to shallow invasion, chronic placental hypoxia, fetal growth restriction, and release of placental injury signals preceding maternal syndrome. Potential failure modes include reduced autophagic flux due to inhibition of autophagosome to lysosome fusion or mistimed persistence of hypoxia signaling, such as prolonged HIF-1α activity. Collectively, this evidence suggests that impaired autophagy is a testable contributor to preeclampsia pathogenesis. Predictions include early risk stratification with circulating autophagy markers and extracellular vesicle microRNAs, and therapeutic benefit from autophagy modulation that targets AMPK or mTOR or activates TFEB with safety constraints. This framework reframes preeclampsia as a disorder of placental quality control and specifies where and when autophagy may be required.

The human maternal-fetal interface represents a unique immunological environment where maternal immune cells and fetal extravilllous trophoblasts (EVT) engage in direct cellular contact to establish and maintain immune tolerance. EVT exhibit a distinct immunophenotype characterized by the expression of tolerogenic molecules, including HLA-G, PD-L1, and PD-L2, which play pivotal roles in modulating maternal immune responses. In addition, human EVT express HLA-C that can be directly recognized by maternal NK and T cells and promotes their activation. Importantly, no orthologues of HLA-C or HLA-G are present in animal models used for research, and murine trophoblast lack MHC expression. Thus, investigating the phenotypic and functional properties of human EVT from placental tissues, under both physiological and pathological conditions, is essential for elucidating the mechanisms underlying pregnancy maintenance and pathophysiology of placental inflammation associated with preeclampsia, preterm birth, fetal growth restriction, and placenta accreta. This protocol outlines a comprehensive methodology for the isolation and characterization of primary human EVT from healthy term placental tissue as well as after preterm birth and preeclampsia. It includes: (1) dissection of placental and chorionic membrane tissues; (2) enzymatic digestion and the preparation of single cell suspensions; (3) EVT purification by FACS sort; (4) Phenotypic characterization by high-dimensional flow cytometry; and (5) short-term in vitro culture and co-culture with maternal immune cells for up to 96 h. Emphasis is placed on obtaining highly pure and viable EVT suitable for downstream applications such as immunological functional assays, protein and gene expression analysis. The implementation of this protocol enables a robust and reproducible platform for advancing understanding of EVT and maternal immune cell interactions and its implications in both healthy and complicated pregnancies.

The human maternal-fetal interface represents a unique immunological environment where maternal immune cells and fetal extravilllous trophoblasts (EVT) engage in direct cellular contact to establish and maintain immune tolerance. EVT exhibit a distinct immunophenotype characterized by the expression of tolerogenic molecules, including HLA-G, PD-L1, and PD-L2, which play pivotal roles in modulating maternal immune responses. In addition, human EVT express HLA-C that can be directly recognized by maternal NK and T cells and promotes their activation. Importantly, no orthologues of HLA-C or HLA-G are present in animal models used for research, and murine trophoblast lack MHC expression. Thus, investigating the phenotypic and functional properties of human EVT from placental tissues, under both physiological and pathological conditions, is essential for elucidating the mechanisms underlying pregnancy maintenance and pathophysiology of placental inflammation associated with preeclampsia, preterm birth, fetal growth restriction, and placenta accreta. This protocol outlines a comprehensive methodology for the isolation and characterization of primary human EVT from healthy term placental tissue as well as after preterm birth and preeclampsia. It includes: (1) dissection of placental and chorionic membrane tissues; (2) enzymatic digestion and the preparation of single cell suspensions; (3) EVT purification by FACS sort; (4) Phenotypic characterization by high-dimensional flow cytometry; and (5) short-term in vitro culture and co-culture with maternal immune cells for up to 96 h. Emphasis is placed on obtaining highly pure and viable EVT suitable for downstream applications such as immunological functional assays, protein and gene expression analysis. The implementation of this protocol enables a robust and reproducible platform for advancing understanding of EVT and maternal immune cell interactions and its implications in both healthy and complicated pregnancies.