Concomitant with the global obesity epidemic, fertility has declined in Western countries with birth rates falling below replacement levels. A higher body mass index (BMI) among women is associated with reduced fertility, but associations with body size earlier in life are underexplored. To investigate whether trajectories of childhood BMI are associated with timing of first childbirth and infertility in women. This prospective, population-based cohort study used linked national register and school health examination data of women aged 18 to 45 years who were born in Copenhagen, Denmark, between 1950 and 1989. Participants were followed up for reproductive outcomes from January 1, 1977 to December 31, 2022. Analytic work was conducted from May to October 2025. Measured height and weight data used to calculate childhood BMI trajectories from ages 6 to 15 years. Registration of childbirth and hospital-based diagnoses of infertility identified in the Medical Birth Register, the Danish Infertility Cohort, the National Patient Register, and the In Vitro Fertilization Register. Hazard ratios (HRs) and 95% CIs were estimated using Cox regression models. This study included 60 864 women in the childbirth analysis, of whom 47 669 (78.3%) gave birth (median [IQR] age, 27.2 [23.7-30.9] years); 58 148 were included in the primary infertility analysis, of whom 5381 (9.3%) were diagnosed with primary infertility (median [IQR] age, 31.3 [27.9-35.1] years); and 63 066 were included in the any infertility analysis, of whom 6961 (11.0%) were diagnosed with any infertility (median [IQR] age, 31.9 [28.4-35.7] years). Women with an obesity trajectory in childhood had a lower hazard of giving birth than those with an average trajectory, and this hazard decreased with increasing age (ages 25-29 years: HR, 0.78 [95% CI, 0.72-0.86]; ages 35-45 years: HR, 0.56 [95% CI, 0.46-0.67]). Similarly, women with an overweight trajectory during childhood had a lower hazard of giving birth, which decreased with increasing age. No associations were observed between the childhood BMI trajectories and primary or any infertility. In this cohort study of Danish women, associations with childbirth decreased from mid- to late-reproductive ages, when the window of fertility narrows. These findings suggest that childhood body size is associated with timing of childbirth, highlighting the importance of addressing women's reproductive health early in the life course.
Fertility patterns among women aged 30 years and above have undergone substantial changes in India, reflecting the country's ongoing demographic transition. However, evidence on the diversity and distribution of age-specific fertility across states and urban-rural settings remains limited. The study examines the diversity-index using age-specific fertility rates (ASFR) among women aged 30-49 years across 16 major Indian states and the Union Territory of Delhi, with particular focus on urban-rural differentials over five rounds of the National Family Health Survey (NFHS) from 1992-93 to 2019-21. Regional and temporal variations in fertility diversity were evident across India. Southern and western states indicates the lower diversity indices, representing more concentrated fertility patterns, whereas northern and eastern states, particularly Bihar, Uttar Pradesh, and Rajasthan, showed greater fertility heterogeneity. Urban areas consistently demonstrated lower fertility diversity than rural areas. Overall, most states experienced a decline in fertility diversity over time, reflecting increasing concentration of childbearing within specific age groups. The findings indicate that fertility beyond age 30 is becoming increasingly concentrated across India, although substantial regional and rural-urban disparities persist. These results highlight the need for region-specific reproductive health and family planning strategies and provide valuable evidence for understanding fertility transitions and informing demographic and women's reproductive health policies.
To identify the specific support needs of Black women navigating infertility care and generate strategies to improve access, patient experience, and outcomes. Mixed-methods observational study SUBJECTS: Black women aged 18-45 years in the US who were currently seeking or had recently sought infertility care EXPOSURE: Online survey and semi-structured interviews MAIN OUTCOME MEASURE: Quantitative data on demographics and fertility care experiences; qualitative data on perceived support needs and recommended solutions RESULTS: 176 women responded to the online survey; of these, 41 women provided consent to participate in semi-structured interviews with the study team. Most participants (74.3%) were 35-45 years of age, married or partnered, held an advanced degree, worked full-time, and had insurance that covered some or all fertility services. 25.5% of participants reported feeling under-supported by friends, family, doctors, or others during their infertility journey, with 31.3% identifying this lack of support as a barrier to initiating treatment. Participants described feelings of loneliness and isolation, with few affirming spaces or communities to find resources and connect with other Black women with infertility. They also shared examples of racism, discrimination, and a lack of compassionate care that led to dismissal of health concerns and delays in referral to reproductive endocrinologists that complicated their infertility journeys. Study participants provided several suggestions for clinics and providers to improve the support of Black women seeking infertility care, from enhancing patient-centered care to increasing access to Black reproductive medicine and mental health providers. Understanding the specific support needs of Black women seeking infertility care will inform patient-centered, compassionate, and culturally responsive care that improves the patient's experience and outcomes.
Hereditary angioedema (HAE) is a rare, potentially life-threatening disorder caused by autosomal dominant mutations in the SERPING1 gene, leading to deficiency or dysfunction of C1 esterase inhibitor. Recent estimates of global HAE prevalence are 1.22 per 100,000 individuals. With total fertility rates (TFRs) decreasing worldwide, several developed countries are now well below the population replacement rate in an "ultra-low fertility" group. With the decrease in fertility, how rare diseases are approached should be re-examined. In this letter, we describe how continued and sustained declines in total fertility rate and population may influence the future number of new HAE cases. We also address disease-specific factors that may further influence future case count, including reproductive hesitancy related to autosomal dominant inheritance, estrogen-associated exacerbation of angioedema attacks, and potential fertility effects of complement dysregulation. These demographic considerations are relevant to clinicians, researchers, and pharmaceutical companies involved in HAE care. Better characterizing population changes in the context of reduced fertility rates may help inform long-term clinical planning and the development of novel therapies for HAE and other rare diseases.
Transgender and gender-diverse (TGD) individuals face significant barriers to fertility preservation, yet existing guidance from ESHRE, ASRM, WPATH, and the Endocrine Society remains too general for translation into implementation-ready national pathways. This review synthesizes evidence on the reproductive effects of gender-affirming hormone therapy, clinical outcomes of fertility preservation in TGD populations, the intersection of neurodiversity and gender diversity, and the structural, interpersonal, and intrapersonal barriers to equitable reproductive care-translating this evidence into a trans-inclusive clinical pathway. We present TransFertility, a dysphoria-informed service model developed at Guy's and St Thomas' NHS Foundation Trust, London. Over 39 months, the model achieved a greater than 19-fold increase in referrals, 100% consultation attendance, over 90% treatment initiation, and 96.7% treatment completion, with no serious adverse events. The absence of a UK national pathway for trans-inclusive fertility preservation represents a critical equity gap. We propose that the evidence synthesized here, together with TransFertility's outcomes, provides a robust foundation for national consensus guideline development. This work's principal contribution is demonstrating that dysphoria-informed care can move from theoretical framework to functioning clinical reality: a reproducible, multidisciplinary pathway showing that structural redesign of administrative, clinical, and psychosocial components improves access, engagement, and treatment completion for TGD individuals.
Oxidative balance within follicular fluid (FF) plays an important role in oocyte competence and female fertility. However, most previous studies have focused on isolated oxidative biomarkers rather than on the coordinated organization of the follicular redox system. This study aimed to identify system-level oxidative phenotypes within FF and evaluate their associations with infertility-related clinical and metabolic factors. Seventy-seven women undergoing IVF treatment were included. Nineteen oxidative and antioxidative biomarkers were measured in FF collected separately from both ovaries. Bayesian multivariable regression with Markov chain Monte Carlo inference was combined with directional indicator analysis and unsupervised taxonomic clustering to identify latent oxidative phenotypes. Two structurally distinct and antagonistically organized oxidative phenotypes were identified within FF. Phenotype 1 comprised biomarkers representing the primary antioxidant defense system, whereas Phenotype 2 included enzymes involved predominantly in secondary detoxification pathways. Idiopathic infertility and BMI demonstrated significant and opposite directional effects across the two phenotypes. Specifically, increasing BMI and idiopathic infertility were associated with decreased activity of biomarkers belonging to Phenotype 1 and increased activity of biomarkers assigned to Phenotype 2 (p = 0.0177 and p = 0.0097, respectively). The follicular redox environment demonstrates a polarized and system-level biochemical architecture rather than uniform oxidative alterations. BMI and idiopathic infertility appear to selectively reorganize oxidative pathways within FF through opposite effects on two antagonistically structured phenotypes. These findings suggest a shared pathophysiological mechanism reorganizing the follicular antioxidant network in both conditions and challenge the single-biomarker paradigm in reproductive oxidative stress research.
Environmental stress is increasingly recognized as an important determinant of reproductive performance in breeding stallions, particularly in the context of rising temperatures, climate variability, and increasingly intensive management systems. Stallions are especially susceptible because spermatogenesis is highly sensitive to temperature and reproductive activity is regulated by seasonal physiological changes. This review critically evaluates the current understanding of the molecular and cellular mechanisms through which environmental stress affects stallion fertility, with particular emphasis on heat stress, oxidative imbalance, endocrine dysregulation, and sperm functional competence. The available evidence indicates that oxidative stress is currently supported by the strongest stallion-specific data linking environmental stress with impaired reproductive function. In contrast, apoptosis, mitochondrial dysfunction, epigenetic regulation, and fertilization-related signaling pathways are supported primarily by mechanistic studies in other mammalian species and remain insufficiently validated under equine-specific conditions. Environmental stress may compromise not only spermatogenesis but also sperm functional competence, including processes required for successful fertilization, potentially resulting in subfertility despite apparently normal conventional semen parameters. Although mechanistic understanding has advanced considerably, important evidence gaps remain because stallion-specific studies are limited and many proposed mechanisms rely on carefully interpreted cross-species evidence. At present, management-based strategies, including optimization of thermal conditions, housing, and breeding management, provide the strongest evidence for mitigating the adverse effects of environmental stress in stallions. By comparison, antioxidant supplementation, nutritional interventions, molecular biomarkers, and other targeted approaches remain promising but require further validation under equine-specific conditions. Future progress will depend on well-designed stallion-specific studies integrating molecular biomarkers, endocrine profiling, thermophysiological assessment, and comprehensive evaluation of sperm functional competence. Such studies will be essential for improving early detection of reproductive dysfunction and for developing evidence-based strategies to maintain stallion fertility under increasingly challenging environmental conditions.
Transcription Factor EB (TFEB) is widely recognized as a key transcription factor regulating lysosomal biogenesis and autophagy. Although the TFEB gene is highly expressed in the testes, the mechanism by which it affects male fertility remains unclear. Here, we report that spermatogonia-specific deletion of TFEB in mice results in a multifaceted reproductive phenotype, including impaired fertility, compromised sperm motility, and attenuated androgen production. Immunofluorescence results showed a significant decrease in the expression levels of TNP1, a marker for spermiogenesis. Under electron microscopy, we observed abnormalities in the mitochondria of the testes and sperm. Integrated transcriptomic and biochemical analyses identified a cluster of mitochondrial-associated genes, including Star, Slc25a48, Gss, and ROMO1, with functional enrichment pinpointing disruptions in steroidogenic flux and calcium homeostasis. In summary, our study identifies TFEB as a pivotal regulator of mitochondrial integrity in the testes, the loss of which drives male subfertility through metabolic and hormonal dysregulation.
Limosilactobacillus reuteri Y15 (LRY15)-derived polyunsaturated fatty acids (PUFAs) restore follicular cell-cell junctions and fertility. LRY15, isolated from the intestine of alginate oligosaccharides (AOS)-dosed mice, improves ovarian function and fertility in cisplatin-induced subfertile mice. LRY15 produces PUFAs, whereas deletion of holo-acyl carrier protein synthase (AcpS) in LRY15 reduces PUFA synthesis and weakens its protective effects. Docosahexaenoic acid (DHA) supplementation recapitulates LRY15-mediated improvements in follicle and embryo development. Stereo-seq analysis reveals that LRY15 restores ovarian cell-cell junctions, suggesting junction remodeling as an important cellular process associated with LRY15 treatment. The Fads2-deficient mouse model further validates that LRY15-derived PUFAs restore follicular junction integrity and improve fertility.
This study examines whether religious coping self-efficacy accounts for the association between religiosity and assisted reproductive technology (ART) acceptance and whether personal infertility experience moderates this indirect association in a highly religious Ghanaian population. A cross-sectional survey of 938 adults in the Greater Accra region of Ghana used multi-stage probability sampling. Generalized additive models tested a dual-stage moderated mediation framework, with bootstrapped confidence intervals for indirect associations. Religiosity showed a negligible direct association with ART acceptance but a positive indirect association through religious coping self-efficacy (ab = 0.031, 95% confidence interval: 0.015 to 0.050). The infertility experience showed compensatory moderation, where the religiosity-to-coping association was weaker among experienced respondents while the coping-to-acceptance association was stronger. Religious coping self-efficacy was more closely associated with acceptance than belief intensity was. Religiosity is related to ART acceptance mainly through religious coping self-efficacy rather than directly. The findings reposition Pargament's religious coping theory as a contextually contingent model. Fertility services might benefit from assessing religious coping capability rather than belief intensity alone.
piRNAs are small regulatory RNAs with critical roles in transposon silencing and regulation of gametogenesis. Phase separation is a driving force for piRNA biogenesis machinery formation; however, the specific piRNA-related proteins that mediate phase separation and how they coordinate to drive piRNA production remain incompletely understood. Through systematic screening, we show here that TDRD5 undergoes phase separation to promote piRNA biogenesis. TDRD5 condensation is mediated by its Lotus domains and an intrinsically disordered region enriched with aromatic residues. Furthermore, TDRD1 and TDRD5 colocalize and synergistically enhance each other's phase separation. TDRD5 phase separation is required for intermitochondrial cement assembly and fetal piRNA processing in fetal/neonatal gonocytes but is dispensable for pachytene piRNA biogenesis in adult mice. TDRD5 phase separation deficiency impairs male fertility in mice and may contribute to human male infertility. Our findings highlight the critical but diverse contributions of phase-separated proteins to germ granule assembly and piRNA production.
Fertility-sparing treatment (FST) is an accepted option for selected women with endometrial carcinoma (EC) or atypical endometrial hyperplasia (AEH) wishing to preserve fertility. However, the impact of FST on subsequent endometrial function remains poorly understood. Oocyte donation (OD) offers a unique model to evaluate endometrial receptivity independently of oocyte quality. We assessed reproductive outcomes after OD in women aged ≥40 years with a history of FST for EC or AEH. This retrospective case series included women aged ≥40 years with histologically confirmed Endometrioid endometrial carcinoma (EEC) or AEH who achieved complete response after FST and subsequently underwent blastocyst transfer through OD. All cycles involved vitrified-warmed donor oocytes, intracytoplasmic sperm injection, blastocyst culture, and elective single embryo transfer. The primary outcome was live birth rate (LBR) per embryo transfer. Sixteen patients underwent 27 embryo transfer cycles. Median age at first transfer was 45.5 years. Embryological outcomes were excellent, with a 96.8% oocyte survival rate and 80.4% top-quality blastocysts. Despite adequate endometrial preparation in all cases, reproductive outcomes were poor. The cumulative LBR per embryo transfer was 18.5% (5/27), while the cumulative LBR per patient was 31.2% (5/16). Patients with disease recurrence showed lower live birth rates than those without recurrence. Endometrial thickness was not associated with implantation outcomes. Women aged ≥40 years undergoing OD after FST for EC or AEH experience reduced reproductive success despite optimal embryo quality. These findings are consistent with the hypothesis of persistent endometrial dysfunction after FST and highlight the need for improved assessment of endometrial receptivity and timely integration of assisted reproductive treatment following complete response.
Psychological resilience is an adaptive capacity that enables individuals to maintain or regain equilibrium following stress. Infertility treatment represents a significant psychological stressor for many couples. Although higher resilience has been associated with better psychological well-being in patients undergoing medically assisted reproduction, its relationship with embryological outcomes remains unknown. This study aimed to determine whether psychological resilience in couples undergoing infertility treatment is associated with blastocyst quality. In this prospective observational study, a total of 126 infertile couples who underwent intracytoplasmic sperm injections, with fresh autologous gametes, were included. Psychological resilience in both partners was assessed using the 10-item Connor-Davidson Resilience Scale. The number of mature oocytes retrieved, semen parameters, and sperm histone retention were evaluated. The primary outcome was the Day 5 good-quality blastocyst development rate. Higher psychological resilience in men, but not in women, was independently associated with a higher good-quality blastocyst development rate (B = 0.06, 95% CI 0.01-0.11, p = 0.02) and remained significant in sensitivity analyses. Male resilience was also positively correlated with sperm morphology (ρ = 0.28, p = 0.001) and inversely correlated with sperm histone retention (ρ = -0.26, p = 0.02). Psychological resilience in men, but not in women, was associated with sperm characteristics and blastocyst quality in couples undergoing infertility treatment. These findings suggest that psychological factors may be reflected in biological parameters relevant to reproduction. Further studies are needed to investigate underlying mechanisms and reproductive outcomes beyond embryo development. Psychological resilience is the ability to adapt to and recover from stressful situations. Infertility treatment can be emotionally challenging for couples, but it is not known whether resilience is related to laboratory outcomes during treatment. In this study, we assessed psychological resilience in 126 couples undergoing intracytoplasmic sperm injection, a fertility treatment in which one sperm is injected into each mature egg. We found that higher resilience in men was associated with better sperm characteristics and a higher rate of good-quality embryos reaching the blastocyst stage on Day 5. This association was not observed in women. These findings suggest that psychological factors in men may be linked to biological features relevant to reproduction. However, this was an observational study and cannot prove cause and effect. Larger studies are needed to confirm these findings and to understand the biological mechanisms involved.
Aligning with the 2026 World Population Day theme that emphasizes "Realizing the Hopes and Aspirations of Young People - Today and for the Future," the Chinese national theme specifically focuses on "Caring for Early Pregnancy, Empowering Healthy Beginnings" (he hu zao yun, guan ai fu neng) to address declining birth rates. Early pregnancy is the critical golden window for preventing birth defects and mitigating maternal risks, yet historical care models disproportionately focus on later gestational stages. This article examines structural gaps in early pregnancy care, including unequal resource distribution, insufficient mental health services, and limited social support networks. These challenges are closely interconnected and collectively limit the effectiveness, accessibility, and continuity of early pregnancy care. To address these challenges, we propose reconstructing a multidimensional collaborative support network. Key strategies include establishing standardized multidisciplinary clinics, leveraging digital health technologies for equitable access, and integrating medical care with supportive national social policies. Strengthening early pregnancy care through coordinated physiological, psychological, and social support can not only improve pregnancy outcomes but also help alleviate fertility anxiety among young couples, contributing to healthier families and high-quality population dynamics during China's demographic transition.
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Artificial intelligence (AI) is increasingly promoted as a transformative innovation in in vitro fertilization (IVF), particularly for embryo selection. This article examines how discourses surrounding AI in IVF reflect broader shifts in medical paradigms and market logics. Drawing on a comparative critical discourse analysis of clinic websites, media texts and professional publications in the Netherlands and the United Kingdom, we explore how stakeholders frame the promise and potential of AI-enabled embryo selection. Our findings reveal three interconnected dynamics. First, UK discourses predominantly construct a paradigm shift from evidence-based medicine (EBM) toward data-driven care, positioning AI as a solution to the perceived limitations of randomised controlled trials. Dutch sources, by contrast, largely sustain EBM principles, framing caution as a safeguard against false hype and escalating costs. Second, UK clinics and technology developers predominantly employ promissory narratives that create self-fulfilling prophecies about an AI-driven future, while Dutch actors largely resist the inevitability of technological fixes. Third, the meaning of patient choice diverges: in the UK, choice is increasingly conflated with consumerism, enabling clinics to market unproven add-ons at additional cost; in the Netherlands, patient choice remains more insulated from market forces, though globalised imaginaries exert growing pressure. These findings illuminate how commercial interests and sociotechnical imaginaries shape reproductive care beyond clinical evidence, raising ethical concerns about equity, regulation and the commodification of hope. The IVF sector offers a critical lens on the wider implications of AI-driven medicine for healthcare governance in an era of marketisation.
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Robot-assisted surgery has been introduced into several male reproductive diseases, including male infertility-related reconstructive or microsurgical procedures, testicular cancer-related retroperitoneal lymph node dissection, and penile cancer-related inguinal lymph node surgery. However, unlike robot-assisted radical prostatectomy, these non-prostate male reproductive applications remain scattered across small clinical series, technical reports, and procedure-specific reviews. This study mapped the global research landscape and thematic evolution of robot-assisted surgery for male reproductive diseases excluding prostate disease. Publications were retrieved from the Web of Science Core Collection using a disease- and procedure-bound search strategy focused on robot-assisted microsurgery, vasovasostomy, vasoepididymostomy, varicocelectomy, sperm retrieval, azoospermia, testicular cancer/RPLND, and penile cancer/inguinal lymphadenectomy. The search was conducted on June 12, 2026. Articles and reviews in English were retained. Bibliometrix and CiteSpace were used to analyze publication trends, countries, institutions, authors, sources, cited references, keyword co-occurrence, clusters, bursts, and thematic changes. A total of 228 records were initially retrieved. After excluding 67 records outside the retained document types and 8 non-English records, 153 publications were included, comprising 110 articles and 43 reviews. The literature covered 2001-2026, involved 59 sources, 952 authors, 298 author keywords, and 3016 cited references, with an annual growth rate of 6.2%. Annual output remained low before 2013, increased after 2019, and peaked in 2025 (n = 18), whereas 2026 represented partial-year data (n = 9). The United States was the dominant contributor and citation center, while Italy, China, India, England, Germany, Canada, France, Switzerland, and Australia formed the main international network. Keyword analysis identified three principal domains: testicular cancer/RPLND, penile cancer/inguinal lymphadenectomy, and male infertility-related robotic microsurgery. Robot-assisted surgery for male reproductive diseases is a small but expanding field organized around distinct oncologic and reproductive surgery applications. Publication and citation activity is more extensive for testicular cancer/RPLND and penile cancer nodal surgery than for fertility-directed microsurgical reconstruction; however, bibliometric prominence should not be interpreted as greater clinical maturity, quality of evidence, or superiority. Future research should emphasize standardized indications, multicenter outcome reporting, fertility and oncologic endpoints, learning-curve assessment, and cost-effectiveness evaluation.
Small size of follicle, which may be due to summer, and high progesterone (P4) could hamper the ovulatory response to gonadotropin-releasing hormone (GnRH) at the beginning of GnRH-based protocols for estrus synchronization (GnRH-1), thus decreasing fertility. Accordingly, the objective of this study was to evaluate the effects of two-day extension in interval between presynchronization (Ovsynch) and breeding (Heatsynch) protocols and administration of prostaglandin F2α (PGF2α) two days before breeding protocol on size of the largest follicle (LF) and P4 concentration (PC) at GnRH-1, respectively, and fertility of cows during summer. Cows were randomly assigned to four experimental groups, including: 1) O7DH (cows with 7-day interval between Ovsynch and Heatsynch; n = 153), 2) O7DH+PG (cows with 7-day interval between Ovsynch and Heatsynch plus administration of PGF2α two days before Heatsynch; n = 156), 3) O9DH (cows with 9-day interval between Ovsynch and Heatsynch; n = 147), and 4) O9DH+PG (cows with 9-day interval between Ovsynch and Heatsynch plus administration of PGF2α two days before Heatsynch; n = 154). Further, cows were subjected to ultrasonographic examination and blood sampling at GnRH-1. Extension of the interval between Ovsynch and Heatsynch did not affect size of LF at GnRH-1 (P > 0.05); however, it decreased pregnancy risk (P < 0.05). Administration of PGF2α two days before Heatsynch decreased PC at GnRH-1 (P < 0.05), but it did not affect pregnancy risk (P > 0.05). In conclusion, the present study revealed that additional two days before breeding protocol did not increase follicle size at GnRH-1 and reduced fertility. Furthermore, PGF2α administration before breeding protocol could not improve fertility of cows despite reducing PC at GnRH-1.
Methionine synthases (MetEs) are essential for plant metabolism and are highly expressed during reproductive processes. Despite their importance, the subcellular localization of MetEs and their specific roles and regulatory mechanisms in plant reproduction remain unclear. To address these knowledge gaps, we conducted subcellular localization and phylogenetic analyses of MetEs in the model species tobacco (Nicotiana tabacum). Additionally, we explored their functions through morphological, metabolomic, and transcriptomic analyses. The results showed that six NtMetEs belonging to dicot subclade 2 (DS2) localized in the endoplasmic reticulum, nucleus and cytosol, while NsylMetE4 of DS1 with a transit peptide targeted to the cytosol and nucleus. Knockout of four NtMetEs abundantly expressed in anthers reduced the pollen germination ratio by an average of 45.15% and methionine (Met) and S-adenosylmethionine (Sam) levels by 32.55% and 47.58%, respectively. Exogenous Met and Sam partially restored pollen germination in mete1234, and Met mainly rescued germination of pollen with normal size both in vivo and in vitro. In mete1234 pollen, sucrose, starch, and energy metabolism were impaired, accompanied by reduced amyloplast accumulation and increased intine deposition. These results indicated that the localization of MetEs was diverse, and the transit peptide did not necessarily determine their plastid localization. NtMetE1-4 synergistically regulated pollen development and germination through modulating carbohydrate metabolism and amyloplast deposition. These findings expand our understanding of MetEs localization, Met synthesis, and pollen fertility regulation, offering implications for plant reproduction.