Whole-genome sequencing of diverse wheat accessions uncovers genetic changes during modern breeding in China and the United States.

Breeding has dramatically changed the plant architecture of wheat (Triticum aestivum), resulting in the development of high-yielding varieties adapted to modern farming systems. However, how wheat breeding shaped the genomic architecture of this crop remains poorly understood. Here, we performed a comprehensive comparative analysis of a whole-genome resequencing panel of 355 common wheat accessions (representing diverse landraces and modern cultivars from China and the United States) at the phenotypic and genomic levels. The genetic diversity of modern wheat cultivars was clearly reduced compared to landraces. Consistent with these genetic changes, most phenotypes of cultivars from China and the United States were significantly altered. Of the 21 agronomic traits investigated, 8 showed convergent changes between the 2 countries. Moreover, of the 207 loci associated with these 21 traits, more than half overlapped with genomic regions that showed evidence of selection. The distribution of selected loci between the Chinese and American cultivars suggests that breeding for increased productivity in these 2 regions was accomplished by pyramiding both shared and region-specific variants. This work provides a framework to understand the genetic architecture of the adaptation of wheat to diverse agricultural production environments, as well as guidelines for optimizing breeding strategies to design better wheat varieties.

Genome sequence of the progenitor of wheat A subgenome Triticum urartu.

Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid (Triticum turgidum, AABB) and hexaploid (Triticum aestivum, AABBDD) wheat1,2. Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping4,5. We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147 T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement.

Dissection and validation of a promising QTL controlling spikelet number on 5B in bread wheat.

KEY MESSAGE: Five environmentally stable QTLs for spikelet number per spike and days to heading were identified using a high-genetic map containing 95,444 SNPs, among which QSns.ucas-5B was validated using residual heterozygous line at multiple environments. Spikelet number per spike (SNS) and days to heading (DTH) play pivotal roles in the improvement of wheat yield. In this study, a high-density genetic map for a recombinant inbred lines (RILs) population derived from Zhengnong 17 (ZN17) and Yangbaimai (YBM) was constructed using 95,444 single-nucleotide polymorphism (SNP) markers from the Wheat660K SNP array. Our study identified a total of five environmentally stable QTLs for SNS and DTH, one of which was named QSns.ucas-5B, with a physical interval of approximately 545.4-552.1 Mb on the 5BL chromosome arm. Importantly, the elite haplotype within QSns.ucas-5B showed a consistent and positive effect on SNS, grain number and weight per spike, without extending the days to heading. These findings provide a foundation for future efforts to map and clone the gene(s) responsible for QSns.ucas-5B and further indicate the potential application of the developed and validated InDel marker of QSns.ucas-5B for molecular breeding purposes, aimed at improving wheat grain yield.

Visible-Light-Promoted α-Benzylation of N-Phenyl α-Amino Acids to α-Amino Phenylpropanoids.

A new method for the synthesis of α-amino phenylpropanoids under blue light-emitting diode irradiation has been developed through α-C-H benzylation of readily available N-phenyl glycine ester with benzyl oxalates as a coupling partner under mild conditions. A range of N-phenyl glycine esters were successfully converted to α-amino phenylpropanoid products in moderate to good yields. The utility of this methodology is underlined by its application to the late-state modification of natural products.

Requirement and functional redundancy of two large ribonucleotide reductase subunit genes for cell cycle, chloroplast biogenesis and photosynthesis in tomato.

BACKGROUND AND AIMS: Ribonucleotide reductase (RNR), functioning in the de novo synthesis of deoxyribonucleoside triphosphates (dNTPs), is crucial for DNA replication and cell cycle progression. In most plants, the large subunits of RNR have more than one homologous gene. However, the different functions of these homologous genes in plant development remain unknown. In this study, we obtained the mutants of two large subunits of RNR in tomato and studied their functions. METHODS: The mutant ylc1 was obtained by ethyl methyl sulfonate (EMS) treatment. Through map-based cloning, complementation and knock-out experiments, it was confirmed that YLC1 encodes a large subunit of RNR (SlRNRL1). The expression level of the genes related to cell cycle progression, chloroplast biogenesis and photosynthesis was assessed by RNA-sequencing. In addition, we knocked out SlRNRL2 (a SlRNRL1 homologue) using CRISPR-Cas9 technology in the tomato genome, and we down-regulated SlRNRL2 expression in the genetic background of slrnrl1-1 using a tobacco rattle virus-induced gene silencing (VIGS) system. KEY RESULTS: The mutant slrnrl1 exhibited dwarf stature, chlorotic young leaves and smaller fruits. Physiological and transcriptomic analyses indicated that SlRNRL1 plays a crucial role in the regulation of cell cycle progression, chloroplast biogenesis and photosynthesis in tomato. The slrnrl2 mutant did not exhibit any visible phenotype. SlRNRL2 has a redundant function with SlRNRL1, and the double mutant slrnrl1slrnrl2 is lethal. CONCLUSIONS: SlRNRL1 is essential for cell cycle progression, chloroplast biogenesis and photosynthesis. In addition, SlRNRL1 and SlRNRL2 possess redundant functions and at least one of these RNRLs is required for tomato survival, growth and development.

Biofortification of iron and zinc in rice and wheat.

Iron and zinc are critical micronutrients for human health. Approximately two billion people suffer from iron and zinc deficiencies worldwide, most of whom rely on rice (Oryza sativa) and wheat (Triticum aestivum) as staple foods. Therefore, biofortifying rice and wheat with iron and zinc is an important and economical approach to ameliorate these nutritional deficiencies. In this review, we provide a brief introduction to iron and zinc uptake, translocation, storage, and signaling pathways in rice and wheat. We then discuss current progress in efforts to biofortify rice and wheat with iron and zinc. Finally, we provide future perspectives for the biofortification of rice and wheat with iron and zinc.

Iron in plant-pathogen interactions.

Iron is an essential element for most organisms. As an indispensable co-factor of many enzymes, iron is involved in various crucial metabolic processes that are required for the survival of plants and pathogens. Conversely, excessive iron produces highly active reactive oxygen species, which are toxic to the cells of plants and pathogens. Therefore, plants and pathogens have evolved sophisticated mechanisms to modulate iron status at a moderate level for maintaining their fitness. Over the past decades, many efforts have been made to reveal these mechanisms, and some progress has been made. In this review, we describe recent advances in understanding the roles of iron in plant-pathogen interactions and propose prospects for future studies.

Glutamate synthase 1 is involved in iron-deficiency response and long-distance transportation in Arabidopsis.

Iron is an essential microelement for plant growth. After uptake from the soil, iron is chelated by ligands and translocated from roots to shoots for subsequent utilization. However, the number of ligands involved in iron chelation is unclear. In this study, we identified and demonstrated that GLU1, which encodes a ferredoxin-dependent glutamate synthase, was involved in iron homeostasis. First, the expression of GLU1 was strongly induced by iron deficiency condition. Second, lesion of GLU1 results in reduced transcription of many iron-deficiency-responsive genes in roots and shoots. The mutant plants revealed a decreased iron concentration in the shoots, and displayed severe leaf chlorosis under the condition of Fe limitation, compared to wild-type. Third, the product of GLU1, glutamate, could chelate iron in vivo and promote iron transportation. Last, we also found that supplementation of glutamate in the medium can alleviate cadmium toxicity in plants. Overall, our results provide evidence that GLU1 is involved in iron homeostasis through affecting glutamate synthesis under iron deficiency conditions in Arabidopsis.

Draft genome of the wheat A-genome progenitor Triticum urartu.

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

A FIT-binding protein is involved in modulating iron and zinc homeostasis in Arabidopsis.

Fe and Zn are essential micronutrients for plant growth, and the interrelationship regarding their homeostasis is very complicated. In this study, we identified a FIT-binding protein (FBP) using the yeast two-hybrid system. The C-terminus of FBP binds to the bHLH domain of FIT, abolishing the DNA-binding capacity of FIT. Knockout of FBP results in an enhanced expression of NAS genes and a higher nicotianamine content, and the fbp mutant exhibits tolerance to excessive Zn. Physiological analyses reveal that the mutant fbp retains a larger amount of Zn in roots and transfers a greater proportion of Fe to shoots than that in wild type under Zn-excessive stress. As FBP is expressed in the root stele, the negative regulation caused by sequestration of FIT is restricted to this tissue, whereas other FIT-regulated genes, such as IRT1 and FRO2, which mainly expressed in root epidermis, do not show transcriptional upregulation in the fbp mutant. As an antagonistic partner, FBP offers a new approach to spatially fine-tune the expression of genes controlled by FIT. In conclusion, our findings provide a new insight to understand the interrelationship of Fe and Zn homeostasis in plants.

Characterization of the AtSPX3 Promoter Elucidates its Complex Regulation in Response to Phosphorus Deficiency.

AtSPX3, responding to phosphate (Pi) deficiency by its expression, is an important gene involved in Pi homeostasis in Arabidopsis. To understand its transcriptional regulation, we characterized the AtSPX3 promoter by distal truncation, internal deletion and mutation of the predicted cis-elements, and identified multiple cis-elements responsive to Pi status. The P1BS (AtPHR-binding site) and AtMyb4 (putative MYB4-binding site) elements were two main cis-elements in the AtSPX3 promoter. P1BS is essential and has a dosage effect for activating expression of the gene under Pi deficiency, while the element AtMyb4 possesses a dual function: one is to enhance AtSPX3 expression in roots under Pi deficiency, and the other one is to repress AtSPX3 expression in shoots under both Pi deficiency and sufficiency. Moreover, we confirmed that AtPHR1, a key transcription factor in Pi homeostasis of plants, was required for the negative regulation function of the AtMyb4 element in shoots. Additionally, we also found that the AtSPX3 promoter had a length limitation for activating gene expression. Generally, our findings in this work are useful for understanding the molecular regulation mechanism of genes involved in Pi uptake and homeostasis.

Prevalence of Wenyonella philiplevinei infection in Linwu ducks in Linwu county, subtropical China.

Wenyonella philiplevinei is one of the pathogens causing coccidiasis in ducks. The prevalence of W. philiplevinei infection in Linwu ducks (a Chinese local domesticated duck) was investigated in Linwu county (an area of Hunan province) of Hunan province, subtropical China from March 2014 to February 2015. Two hundred fifty-eight of 1800 (14.3 %) Linwu ducks were found to be infected with W. philiplevinei. To identify the species identity of the collected samples, a portion of the 18S rDNA was amplified from W. philiplevinei by polymerase chain reaction (PCR) and cloned and then sequenced. The sequences of 18S rDNA of all samples were 422 bp in size. The A + T content of the 18S rDNA sequences is 58-59 %. Sequence comparison revealed that the similarity in 18S rDNA sequences among Hunan isolates with that of the W. philiplevinei available (Guangdong isolate) were more than 98 %. The intra-specific sequence variations within each of the Hunan isolates were 0-1.7 %. Phylogenetic analysis using maximum likelihood (ML) method indicated that the genus Wenyonella is more closely related to Eimeria + Cyclospora than to that of the Isospora. These new data provide a genetic marker for the differentiation of W. philiplevinei or other closely related coccidian. This is the first report of W. philiplevinei prevalence in ducks in Hunan province, subtropical China.

[Safety of the offspring conceived by assisted reproductive technology with cryopreserved donor sperm].

OBJECTIVE: To investigate the pregnancy outcomes of assisted reproductive technology (ART) with cryopreserved donor sperm and the safety of the offspring thus conceived. METHODS: The Human Sperm Bank of CITIC Xiangya Hospital provided cryopreserved donor semen to 31 reproductive centers in China between January 2006 and December 2012, with which 50247 ART cycles were accomplished. We compared the rates of birth defects and spontaneous abortion of intracervical insemination (ICI), intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). RESULTS: A total of 39 047 ART cycles were performed by artificial insemination with cryopreserved donor sperm, including 36 674 cycles of ICI and 2 372 cycles of IUI. Among the 8 612 clinical pregnancies achieved by ICI, there were 917 cases of spontaneous abortion (at <28 gestational wk) (10.6%) and 6133 live births, with 43 cases of birth defect (0.70%). Of the 547 clinical pregnancies achieved by IUI, there were 41 cases of spontaneous abortion (7.5%) and 426 live births, with 2 cases of birth defect (0.47%). Totally, 11 200 cycles of IVF and ICSI were accomplished with cryopreserved donor sperm. Of the 5 860 clinical pregnancies achieved by IVF, there were 456 cases of spontaneous abortion (7.8%) and 5089 live births, with 55 cases of birth defect (1.08%). Among the 350 clinical pregnancies achieved by ICSI, there were 30 cases of spontaneous abortion (8.6%) and 229 live births, with 3 cases of birth defect (1.31%). The birth defect rate of ART with cryopreserved donor sperm was significantly lower than that published by the Chinese Ministry of Health (0.86% vs 1.53%,P<0.01). CONCLUSIONS: The safety of the offspring conceived by ART with cryopreserved donor sperm is controllable.

SKB1/PRMT5-mediated histone H4R3 dimethylation of Ib subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis thaliana.

Histone modifications play critical roles in the perception of environmental cues by plants. Here, we report that Shk1 binding protein 1 (SKB1/AtPRMT5), which catalyzes the symmetric dimethylation of histone H4R3 (H4R3sme2), is involved in iron homeostasis in Arabidopsis. The SKB1 lesion mutant exhibited higher iron accumulation in shoots and greater tolerance to iron deficiency than the wild type. The expression of SKB1 was not affected by iron, but the level of H4R3sme2 mediated by SKB1 was related to iron status in plants. We showed by chromatin immunoprecipitation (ChIP) and genome-wide ChIP-seq that SKB1 associated with the chromatin of the Ib subgroup bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101), and symmetrically dimethylated histone H4R3. The quantity of SKB1 that associated with chromatin of the Ib subgroup bHLH genes and the level of H4R3sme2 corresponded to the iron status of plants (higher with increased iron supply and lower when iron was removed). We conclude that SKB1-mediated H4R3sme2 regulates iron homeostasis in Arabidopsis in the context of increasing or decreasing expression of Ib subgroup bHLH genes. Iron deficiency may cause an increase in the disassociation of SKB1 from chromatin of the bHLH genes and a decrease in the level of H4R3sme2, thereby elevating their transcription and enhancing iron uptake. Our findings provide new insight into the molecular mechanisms of iron homeostasis in strategy I plants.

SlbHLH068 interacts with FER to regulate the iron-deficiency response in tomato.

BACKGROUND AND AIMS: Iron is an essential micronutrient for all organisms and its uptake, translocation, distribution and utilization are regulated in a complex manner in plants. FER, isolated from tomato (Solanum lycopersicum), was the first transcription factor involved in the iron homeostasis of higher plants to be identified. A FER defect in the T3238fer mutant drastically downregulates the expression of iron uptake genes, such as ferric-chelate reductase 1 (LeFRO1) and iron-regulated transporter 1 (LeIRT1); however, the molecular mechanism by which FER regulates genes downstream remains unknown. The aim of this work was therefore to identify the gene that interacts with FER to regulate the iron-deficiency response in tomato. METHODS: The homologue of the Arabidopsis Ib subgroup of the basic helix-loop-helix (bHLH) proteins, SlbHLH068, was identified by using the program BLASTP against the AtbHLH39 amino acid sequence in the tomato genome. The interaction between SlbHLH068 and FER was detected using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. In addition, virus-induced gene silencing (VIGS) was used to generate tomato plants in which SlbHLH068 expression was downregulated. The expression of genes was analysed using northern blot hybridization and multiple RT-PCR analysis. Seedlings of wild-type and mutant plants were grown under conditions of different nutrient deficiency. KEY RESULTS: SlbHLH068 is highly upregulated in roots, leaves and stems in response to iron deficiency. An interaction between SlbHLH068 and FER was demonstrated using yeast two-hybrid and BiFC assays. The heterodimer formed by FER with SlbHLH068 directly bound to the promoter of LeFRO1 and activated the expression of its reporter gene in the yeast assay. The downregulation of SlbHLH068 expression by VIGS resulted in a reduction of LeFRO1 and LeIRT1 expression and iron accumulation in leaves and roots. CONCLUSIONS: The results indicate that SlbHLH068, as a putative transcription factor, is involved in iron homeostasis in tomato via an interaction with FER.

Mediator subunit 16 functions in the regulation of iron uptake gene expression in Arabidopsis.

Iron is an essential nutrient for plant growth and development, and its absorption is tightly controlled. Under iron limitation, FIT dimerizes with the four Ib bHLH proteins and activates the expression of iron uptake genes. However, how the dimerized complex activates downstream genes remains unclear. Using forward genetics, a low-iron-sensitive mutant was screened. The corresponding gene (MED16) was isolated, and its biological functions in iron homeostasis were characterized using approaches such as gene expression, protein subcellular localization, protein-protein interaction and chromatin immunoprecipitation assay. Lesion of MED16 significantly reduced FRO2 and IRT1 expression in Arabidopsis roots. The MED16 mutants showed a low shoot iron concentration and severe leaf chlorosis under iron limitation, whereas it grew normally as wild-type under iron sufficiency. Furthermore, we showed that MED16 interacted with FIT and improved the binding of the FIT/Ib bHLH complex to FRO2 and IRT1 promoters under iron-deficient conditions. Additionally, we found that many iron-deficient response genes, which are regulated by FIT, were also controlled by MED16. In conclusion, MED16 is involved in the iron deficiency response, and modulates the iron uptake gene expression under iron limitation. Our results increase the understanding of the molecular regulation mechanisms underlying iron uptake and homeostasis in plants.

Zebrafish rgs4 is essential for motility and axonogenesis mediated by Akt signaling.

The schizophrenia susceptibility gene, Rgs4, is one of the most intensively studied regulators of G-protein signaling members, well known to be fundamental in regulating neurotransmission. However, little is known about its role in the developing nervous system. We have isolated zebrafish rgs4 and shown that it is transcribed in the developing nervous system. Rgs4 knockdown did not affect neuron number and patterning but resulted in locomotion defects and aberrant development of axons. This was confirmed using a selective Rgs4 inhibitor, CCG-4986. Rgs4 knockdown also attenuated the level of phosphorylated-Akt1, and injection of constitutively-activated AKT1 rescued the motility defects and axonal phenotypes in the spinal cord but not in the hindbrain and trigeminal neurons. Our in vivo analysis reveals a novel role for Rgs4 in regulating axonogenesis during embryogenesis, which is mediated by another schizophrenia-associated gene, Akt1, in a region-specific manner.

[Qualitative and quantitative diagnostic performance of 320-slice computed tomography for detecting coronary artery disease with respect to atherosclerotic plaque characteristics].

OBJECTIVE: To investigate qualitatively and quantitatively the diagnostic performance of 320-slice CT for detection of coronary artery disease with respect to different atherosclerotic plaque characteristics. METHODS: A retrospective search was performed for inpatients underwent both coronary CT and further coronary angiography (CAG) from December 1, 2008 to December 31, 2012. The diagnostic performance of 320-slice CTA for detecting significant stenosis ( ≥ 50% diameter) with respect to atherosclerotic plaque characteristics were analyzed by calculating sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, kappa index (κ), and area under the receiver operating characteristic curve (AUC). Chi-square test was used to evaluate whether there were significant differences of the true-case frequency (true positive + true negative) and false-case frequency (false positive + false negative) among groups. Bland-Altman analysis was used to determine limits of agreement between CTA and CAG. RESULTS: A total of 454 patients and 6 779 segments were analyzed. Diagnostic accuracy was higher in non-calcified segments; whereas they decreased in the presence of both mild-moderately and heavily calcified plaques. Excellent agreement (κ = 0.810) between CT and CAG was observed for non-calcified segments, while good agreement was observed for both mild-moderately (κ = 0.701) and heavily calcified segments (κ = 0.750). Both mild-moderate (P = 0.000) and heavy (P = 0.000) calcification decreased the true-case frequency and increased the false-case frequency when compared to non-calcification. There were no significant underestimation or overestimation for non-calcified (P = 0.087) and mild-moderately calcified (P = 0.704) segments, while there was significant overestimation for heavily calcified segments (P = 0.001). CONCLUSIONS: Great qualitative and quantitative diagnostic performances of 320-slice CT were observed in non-calcified coronary segments. However, qualitative diagnostic performance decreased in both mild-moderately and heavily calcified segments, and quantitative overestimation were observed in heavily calcified segments.

How do HCN channels play a part in Alzheimer's and Parkinson's disease?

Neurodegenerative diseases like Alzheimer's and Parkinson's disease (AD and PD) are well-known, yet their underlying causes remain unclear. Recent studies have suggested that disruption of ion channels contribute to their pathogenesis. Among these channels, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, encoded by HCN1-4 genes, are of particular interest due to their role in generating hyperpolarization-activated current (Ih), which is crucial in various neural activities impacting memory and motor functions. A growing body of evidence underscores the pivotal role of HCN in Aß generation, glial cell function, and ischemia-induced dementia; while HCN is expressed in various regions of the basal ganglia, modulating their functions and influencing motor disorders in PD; neuroinflammation triggered by microglial activation represents a shared pathological mechanism in both AD and PD, in which HCN also plays a significant part. This review delves into the neuronal functions governed by HCN, its roles in the aforementioned pathogenesis, its expression patterns in AD and PD, and discusses potential therapeutic drugs targeting HCN for the treatment of these diseases, aiming to offer a novel perspective and inspire future research endeavors.

Long-term storage modifies the microRNA expression profile of cryopreserved human semen.

The global practice of cryopreservation of human semen is commonplace in Assisted Reproductive Technology (ART) labs and sperm banks. However, information on the effects of long-term cryopreservation on semen is limited to clinical data summaries and descriptions. For this study, we prepared 4 semen specimens of fresh semen, 4 specimens cryostored for at least 1 year, 3 specimens cryostored for at least 5 years, 4 specimens cryostored for at least 10 years, and 3 specimens cryostored for at least 15 years. Total RNA was extracted from each sample, amplified, labeled, and mapped to the known primary microRNA (miRNA) in the miRBase database, enabling the prediction of novel miRNAs. We found that cryopreservation can lead to changes in miRNA expression, and with the increase in storage time, these changes became more pronounced. Meanwhile, the expression of let-7d-3p, let-7c-5p and let-7i-3p miRNAs changed dynamically over cryostorage time in frozen-thawed human sperm. Furthermore, we analyzed the time-dependent dynamics of cryostorage-expressed miRNAs and their target mRNAs and found that half of the target genes were expressed in oocytes. These intersection genes were mainly enriched in cancer and cytoskeletal signaling pathways. Our findings showed that the miRNA expression profile of cryopreserved human semen is modified by long-term storage. Furthermore, as the storage time increases, the impact on human sperm becomes more pronounced in terms of miRNAs, which may have an effect on subsequent fertilization and embryonic development.