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Age-related osteoporosis is a metabolic skeletal disorder caused by estrogen deficiency in postmenopausal women. Prolonged use of anti-osteoporotic drugs such as bisphosphonates and FDA-approved anti-resorptive selective estrogen receptor modulators (SERMs) has been associated with various clinical drawbacks. We recently discovered a low-molecular-weight biocompatible and osteoanabolic phytoprotein, called HKUOT-S2 protein (32 kDa), from Dioscorea opposita Thunb that can accelerate bone defect healing. Here, we demonstrated that the HKUOT-S2 protein treatment can enhance osteoblasts-induced ossification and suppress osteoporosis development by upregulating skeletal estrogen receptors (ERs) ERα, ERβ, and GPR30 expressions in vivo . Also, HKUOT-S2 protein estrogenic activities promoted hMSCs-osteoblasts differentiation and functions by increasing osteogenic markers, ALP, and RUNX2 expressions, ALP activity, and osteoblast biomineralization in vitro . Fulvestrant treatment impaired the HKUOT-S2 protein-induced ERs expressions, osteoblasts differentiation, and functions. Finally, we demonstrated that the HKUOT-S2 protein could bind to ERs to exert osteogenic and osteoanabolic properties. Our results showed that the biocompatible HKUOT-S2 protein can exert estrogenic and osteoanabolic properties by positively modulating skeletal estrogen receptor signaling to promote ossification and suppress osteoporosis. Currently, there is no or limited data if any, on osteoanabolic SERMs. The HKUOT-S2 protein can be applied as a new osteoanabolic SERM for osteoporosis treatment., Competing Interests: Kelvin Wai Kwok Yeung is an associate editor and Wei Qiao an editorial board member for Bioactive Materials and were not involved in the editorial review or the decision to publish this article. The authors declare that they have no conflict of interest., (© 2024 The Authors.)

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter., (© 2024. The Author(s).)

Objective: To evaluate the attachment rate of a human embryonic stem cell-derived trophoblastic spheroid onto endometrial epithelial cells in predicting the cumulative live birth rate of an in vitro fertilization (IVF) cycle., Design: A prospective observational study., Setting: University hospital and research laboratory., Patient(s): A total of 240 infertile women from 2017-2021., Intervention(s): Infertile women with regular cycles attending for IVF were recruited. An endometrial aspirate was collected from a natural cycle 1 month before IVF to determine the BAP-EB attachment rate., Main Outcome Measure(s): Cumulative live birth rates from a stimulated cycle and its derived frozen embryo transfer cycles within 6 months of ovarian stimulation were obtained., Result(s): The BAP-EB attachment rate in women who attained a cumulative live birth was similar to that in those who did not. When women were stratified by age into <35 years and ≥35 years, the BAP-EB attachment rate was significantly higher only in women aged ≥35 years having a live birth when compared with those in the same age group without a live birth. Receiver operating characteristic curve analysis of BAP-EB attachment rate in predicting cumulative live birth showed the areas under the curve of 0.559 (95% confidence interval [CI], 0.479-0.639), 0.448 (95% CI, 0.310-0.585), and 0.613 (95% CI, 0.517-0.710) for all ages, an age of <35 years, and an age of ≥35 years, respectively., Conclusion(s): The BAP-EB attachment rate offers only a very modest prediction of the cumulative live birth rate in women aged ≥35 years undergoing IVF., Clinical Trial Registration Number: NCT02713854 (https://clinicaltrials.gov/ct2/show/NCT02713854; Date of registration, March 21, 2016; date of enrollment of the first subject, August 1, 2017)., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Mammalian female reproduction requires a functional ovary. Competence of the ovary is determined by the quality of its basic unit-ovarian follicles. A normal follicle consists of an oocyte enclosed within ovarian follicular cells. In humans and mice, the ovarian follicles are formed at the foetal and the early neonatal stage respectively, and their renewal at the adult stage is controversial. Extensive research emerges recently to produce ovarian follicles in-vitro from different species. Previous reports demonstrated the differentiation of mouse and human pluripotent stem cells into germline cells, termed primordial germ cell-like cells (PGCLCs). The germ cell-specific gene expressions and epigenetic features including global DNA demethylation and histone modifications of the pluripotent stem cells-derived PGCLCs were extensively characterized. The PGCLCs hold potential for forming ovarian follicles or organoids upon cocultured with ovarian somatic cells. Intriguingly, the oocytes isolated from the organoids could be fertilized in-vitro . Based on the knowledge of in-vivo derived pre-granulosa cells, the generation of these cells from pluripotent stem cells termed foetal ovarian somatic cell-like cells was also reported recently. Despite successful in-vitro folliculogenesis from pluripotent stem cells, the efficiency remains low, mainly due to the lack of information on the interaction between PGCLCs and pre-granulosa cells. The establishment of in-vitro pluripotent stem cell-based models paves the way for understanding the critical signalling pathways and molecules during folliculogenesis. This article aims to review the developmental events during in-vivo follicular development and discuss the current progress of generation of PGCLCs, pre-granulosa and theca cells in-vitro ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Chen, Yeung and Lee.)

Human expanded potential stem cells (hEPSC) have been derived from human embryonic stem cells and induced pluripotent stem cells. Here direct derivation of hEPSC from human pre-implantation embryos is reported. Like the reported hEPSC, the embryo-derived hEPSC (hEPSC-em) exhibit a transcriptome similar to morula, comparable differentiation potency, and high genome editing efficiency. Interestingly, the hEPSC-em show a unique H3 lysine-4 trimethylation (H3K4me3) open chromatin conformation; they possess a higher proportion of H3K4me3 bound broad domain (>5 kb) than the reported hEPSC, naive, and primed embryonic stem cells. The open conformation is associated with enhanced trophoblast differentiation potency with increased trophoblast gene expression upon induction of differentiation and success in derivation of trophoblast stem cells with bona fide characteristics. Hippo signaling is specifically enriched in the H3K4me3 broad domains of the hEPSC-. Knockout of the Hippo signaling gene, YAP1 abolishes the ability of the embryo-derived EPSC to form trophoblast stem cells., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Despite advances in in vitro fertilization (IVF), there is still a lack of non-invasive and reliable biomarkers for selecting embryos with the highest developmental and implantation potential. Recently, small non-coding RNAs (sncRNAs) have been identified in biological fluids, and extracellular sncRNAs are explored as diagnostic biomarkers in the prediction of IVF outcomes. To determine the predictive role of sncRNAs in embryo quality and IVF outcomes, a systematic review and meta-analysis was performed. Articles were retrieved from PubMed, EMBASE, and Web of Science from 1990 to 31 July 2022. Eighteen studies that met the selection criteria were analyzed. In total, 22 and 47 different sncRNAs were found to be dysregulated in follicular fluid (FF) and embryo spent culture medium (SCM), respectively. MiR-663b , miR-454 and miR-320a in FF and miR-20a in SCM showed consistent dysregulation in two different studies. The meta-analysis indicated the potential predictive performance of sncRNAs as non-invasive biomarkers, with a pooled area under curve (AUC) value of 0.81 (95% CI 0.78, 0.844), a sensitivity of 0.79 (95% CI 0.72, 0.85), a specificity of 0.67 (95% CI 0.52, 0.79) and a diagnostic odds ratio (DOR) of 8 (95% CI 5, 12). Significant heterogeneity was identified among studies in sensitivity (I2  =  46.11%) and specificity (I2  =  89.73%). This study demonstrates that sncRNAs may distinguish embryos with higher developmental and implantation potentials. They can be promising non-invasive biomarkers for embryo selection in ART. However, the significant heterogeneity among studies highlights the demand for prospective multicenter studies with optimized methods and adequate sample sizes in the future.

Chronic opioid use disorder patients often also use other substances such as amphetamines. The gene-based analysis method was applied in the genomic database obtained from our previous study with 343 methadone maintenance treatment (MMT) patients. We found that the gene encoding gamma-aminobutyric acid type A receptors (GABA-A receptor) delta subunit isoforms ( GABRD ) was associated with amphetamine use in heroin dependent patients under MMT in Taiwan. A total of 15% of the 343 MMT patients tested positive for amphetamine in the urine toxicology test. Two genetic variants in the GABRD , rs2889475 and rs2376805, were found to be associated with the positive urine amphetamine test. They are located in the exon 1 of the splice variant and altered amino acid compositions (T126I, C/T, for rs2889475, and R252Q, G/A, for rs2376805). The CC genotype carriers of rs2889475 showed a four times higher risk of amphetamine use than those with TT genotype. The GG genotype carriers of rs2376805 showed a three times higher risk of amphetamine use than the AA genotype carriers. To our knowledge, this is the first report that demonstrated an association of the delta splice variant isoform in the GABA-A receptor with an increased risk of amphetamine use in MMT patients. Our results suggest that rs2889475 and rs2376805 may be indicators for the functional role and risk of amphetamine use in MMT patients.

Methadone is a synthetic opioid used for the maintenance treatment (MMT) of heroin dependence. It primarily binds to the μ-opioid receptor (MOR; with its gene, namely OPRM1). Methadone is also an N-methyl-D-aspartate (NMDA) receptor antagonist. The role of NMDA receptor in the regulatory mechanisms of methadone dosage in heroin dependent patients is so far not clear. D-amino acid oxidase (DAO) is an important enzyme that indirectly activates the NMDA receptor through its effect on the D-serine level. To test the hypothesis that genetic polymorphisms in the DAO gene are associated with methadone treatment dose and responses, we selected four single nucleotide polymorphisms (SNPs) in DAO from the literature reports of the Taiwanese population. SNPs were genotyped in 344 MMT patients. In this study, we identified a functional SNP rs55944529 in the DAO gene that reveals a modest but significant association with the methadone dosage in the recessive model of analysis (P = 0.003) and plasma concentrations (P = 0.003) in MMT patients. However, it did not show association with plasma methadone concentration in multiple linear regression analysis. It is also associated with the methadone adverse reactions of dry mouth (P = 0.002), difficulty with urination (P = 0.0003) in the dominant model, and the withdrawal symptoms of yawning (P = 0.005) and gooseflesh skin (P = 0.004) in the recessive model. Our results suggest a role of the indirect regulatory mechanisms of the NMDA reporter, possibly via the DAO genetic variants, in the methadone dose and some adverse reactions in MMT patients., (© 2021. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

The prevalence of type 2 diabetes (T2D) is rapidly increasing across the globe. Fetal exposure to maternal diabetes was correlated with higher prevalence of impaired glucose tolerance and T2D later in life. Previous studies showed aberrant DNA methylation patterns in pancreas of T2D patients. However, the underlying mechanisms remained largely unknown. We utilized human embryonic stem cells (hESC) as the in vitro model for studying the effects of hyperglycemia on DNA methylome and early pancreatic differentiation. Culture in hyperglycemic conditions disturbed the pancreatic lineage potential of hESC, leading to the downregulation of expression of pancreatic markers PDX1 , NKX6-1 and NKX6-2 after in vitro differentiation. Genome-wide DNA methylome profiling revealed over 2000 differentially methylated CpG sites in hESC cultured in hyperglycemic condition when compared with those in control glucose condition. Gene ontology analysis also revealed that the hypermethylated genes were enriched in cell fate commitment. Among them, NKX6-2 was validated and its hypermethylation status was maintained upon differentiation into pancreatic progenitor cells. We also established mouse ESC lines at both physiological glucose level (PG-mESC) and conventional hyperglycemia glucose level (HG-mESC). Concordantly, DNA methylome analysis revealed the enrichment of hypermethylated genes related to cell differentiation in HG-mESC, including Nkx6-1 . Our results suggested that hyperglycemia dysregulated the epigenome at early fetal development, possibly leading to impaired pancreatic development.

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1 , SIRT1 and PUMA . They function through transcription regulation of downstream targets ( P53 , CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.

Objective: To study the use of human embryonic stem cell-derived trophoblastic spheroids (BAP-EB) as human blastocyst surrogates for studying early implantation and trophoblast development., Design: Laboratory study., Setting: University research laboratory., Patient(s): Infertile in vitro fertilization patients donating endometrial aspirates and human embryonic stem cells (hESCs: VAL3 and H9/WA09)., Intervention(s): In BAP-EB derived from hESC, transcriptomes analyzed by next-generation RNA sequencing, effects of Hippo signaling pathway studied by a YAP inhibitor, comparison of attachment of BAP-EB onto primary endometrial epithelial cells (EEC) collected at prereceptive and receptive phases, and antibody blocking assay used to study the molecule(s) involved in BAP-EB attachment., Main Outcome Measure(s): Gene expression profiles and endometrial cell attachment rates., Result(s): The BAP-EB differentiation protocol for VAL3 could be used to induce trophoblast differentiation in another hESC line, H9. Transcriptomic analysis showed that the epiblast signature gene expression was reduced while that of the trophoblast was induced during BAP-EB differentiation. Specifically, trophectoderm signature genes were induced in BAP-EB at 48 hours and 72 hours after induction of differentiation. The Hippo signaling pathway was one of the pathways induced during BAP-EB differentiation, and YAP1 inhibitor statistically significantly reduced attachment, outgrowth, and trophoblast gene expressions of BAP-EB. A statistically significantly higher number of BAP-EB derived from both VAL3 and H9 attached onto receptive EEC than prereceptive EEC. The antibody blocking assay demonstrated that endometrial E-cadherin might be critical in early implantation., Conclusion(s): The data suggest that BAP-EB possesses a trophectoderm-like signature, which supports the use of BAP-EB as a blastocyst surrogate for the study of trophoblast development and endometrial receptivity., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Methadone is a synthetic opioid used as maintenance treatment for patients addicted to heroin. Skin irritation is one of the adverse events caused by opioid use. 344 methadone maintenance treatment (MMT) patients were recruited with records and measurements on methadone dose, plasma methadone concentrations, and treatment emergent symptom scales (TESS). 15 patients reported with skin irritation. Five SNPs located within the NECTIN4 genetic region were genotyped. The NECTIN4 gene within the adherens junction interaction pathway was associated with methadone dose in pathway-based genome wide association analyses (P = 0.0008). Three highly-linked SNPs, rs11265549, rs3820097, and rs4656978, were significantly associated with methadone dose (P = 0.0003), plasma concentrations of R,S-methadone (P = 0.0004) and TNF-α (P = 0.010) in all 344 MMT patients, and with self-report skin irritation symptom scores (P = 0.010) in the 15 MMT patients who reported with skin irritation. To identify the possible roles of plasma level of Nectin-4 in the responses to MMT and opioid use, additional age- and gender-matched 51 controls and 83 methadone-free abstinent former heroin users were recruited. Plasma level of Nectin-4 was the highest in MMT patients among the three groups. The results suggest involvement of genetic variants on NECTIN4 in methadone dose. Plasma Nectin-4 level is likely an indicator for continued use of opioids., Competing Interests: The authors have declared that no competing interests exist.

Sirt1 facilitates the reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs). It is regulated by micro-RNA and reported to be a target of miR-135a. However, their relationship and roles on cellular reprogramming remain unknown. In this study, we found negative correlations between miR-135a and Sirt1 during mouse embryonic stem cells differentiation and mouse embryonic fibroblasts reprogramming. We further found that the reprogramming efficiency was reduced by the overexpression of miR-135a precursor but induced by the miR-135a inhibitor. Co-immunoprecipitation followed by mass spectrometry identified 21 SIRT1 interacting proteins including KU70 and WRN, which were highly enriched for DNA damage repair. In accordance, Sirt1 activator resveratrol reduced DNA damage during the reprogramming process. Wrn was regulated by miR-135a and resveratrol partly rescued the impaired reprogramming efficiency induced by Wrn knockdown. This study showed Sirt1 , being partly regulated by miR-135a, bound proteins involved in DNA damage repair and enhanced the iPSCs production.

Delta opioid receptor (DOR) is well known to be involved in heroin dependence. This study tested the hypothesis that single nucleotide polymorphisms (SNPs) in the opioid receptor delta 1 (OPRD1) gene coding region are associated with treatment responses in a methadone maintenance therapy (MMT) cohort in Taiwan. Three hundred forty-four MMT patients were recruited. Diastolic/systolic blood pressure, heart rate, methadone dosage, and plasma concentrations of methadone were recorded. Twenty-five SNPs located within the OPRD1 genetic region were selected and genotyped from the genomic DNA of all 344 participants. After pairwise tagger analyses, tagger SNP rs204047 showed a significant association with methadone dosage (P = 0.0019), and tagger SNPs rs204047 and rs797397 were significantly associated with plasma R, S-methadone concentrations (P < 0.0006) in patients tested negative in the urine morphine test, which indicated patients with a better response to MMT. The major genotype carriers showed a higher methadone dosage and higher plasma concentrations of R, S-methadone than the minor genotype carriers. The results indicated that OPRD1 genetic variants were associated with methadone dosage and methadone plasma concentration in MMT patients with a negative morphine test result.

The recent opioid crisis is one of the rising challenges in the history of modern health care. New and effective treatment modalities with less adverse effects to alleviate and manage this modern epidemic are critically needed. The FDA has recently approved two non-invasive electrical nerve stimulators for the adjunct treatment of symptoms of acute opioid withdrawal. These devices, placed behind the ear, stimulate certain cranial nerves with auricular projections. This neural stimulation reportedly generates a prompt effect in terms of alleviation of withdrawal symptoms resulting from acute discontinuation of opioid use. Current experimental evidence indicates that this type of non-invasive neural stimulation has excellent potential to supplement medication assisted treatment in opioid detoxification with lower side effects and increased adherence to treatment. Here, we review current findings supporting the use of non-invasive neural stimulation in detoxification from opioid use. We briefly outline the neurophysiology underlying this approach of auricular electrical neural stimulation and its role in enhancing medication assisted treatment in treating symptoms of opioid withdrawal. Considering the growing deleterious impact of addictive disorders on our society, further studies on this emerging treatment modality are warranted., Competing Interests: Competing interestsA.C.H.C, I.S.Q, K.J.T, T.D.C, and V.A.P are employees of Northwell Health. K.J.T, T.D.C, and V.A.P are employees of the Feinstein Institutes for Medical Research. K.J.T is Editor in Chief of Bioelectronic Medicine. V.A.P is Executive Editor of Bioelectronic Medicine., (© The Author(s) 2020.)

Animal and epidemiological studies demonstrated association of persistent exposure of TCDD, an endocrine disrupting chemical, to susceptibility of type 2 diabetes (T2D). High doses of TCDD were commonly employed in experimental animals to illustrate its diabetogenic effects. Data linking the epigenetic effects of low doses of TCDD on embryonic cells to T2D susceptibility risks is very limited. To address whether low dose exposure to TCDD would affect pancreatic development, hESCs pretreated with TCDD at concentrations similar to human exposure were differentiated towards pancreatic lineage cells, and their global DNA methylation patterns were determined. Our results showed that TCDD-treated hESCs had impaired pancreatic lineage differentiation potentials and altered global DNA methylation patterns. Four of the hypermethylated genes (PRKAG1, CAPN10, HNF-1B and MAFA) were validated by DNA bisulfite sequencing. PRKAG1, a regulator in the AMPK signaling pathway critical for insulin secretion, was selected for further functional study in the rat insulinoma cell line, INS-1E cells. TCDD treatment induced PRKAG1 hypermethylation in hESCs, and the hypermethylation was maintained after pancreatic progenitor cells differentiation. Transient Prkag1 knockdown in the INS-1E cells elevated glucose stimulated insulin secretions (GSIS), possibly through mTOR signaling pathway. The current study suggested that early embryonic exposure to TCDD might alter pancreatogenesis, increasing the risk of T2D., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: There is no countable biomarker for opioid dependence treatment responses thus far. In this study, we recruited Taiwanese methadone maintenance treatment patients to search for genes involving the regulatory mechanisms of methadone dose by genome-wide association analyses., Methods: A total of 344 Taiwanese methadone maintenance treatment patients were included in a genome-wide association study. The involvement of GRK5 in opioid dependence was then further confirmed by gene expression study on lymphoblastoid cell lines derived from 3 independent age- and gender-matched groups: methadone maintenance treatment patients, medication-free former heroin abusers, and normal controls., Results: The results indicated that GRK5, the gene encoding an enzyme related to μ-opioid receptor desensitization, is associated with methadone dose by additive model of gene-based association analysis (P=6.76×10-5). We found that 6 of the 55 single nucleotide polymorphisms from the genome-wide genotype platform and 2 single nucleotide polymorphisms from the 29 additionally selected single nucleotide polymorphisms were significantly associated with methadone maintenance dose in both genotype and allele type (P ≤ .006), especially in patients who tested negative in the urine morphine test. The levels of GRK5 gene expression were similar between methadone maintenance treatment patients and medication-free former heroin abusers. However, the normal controls showed a significantly lower level of GRK5 gene expression than the other groups (P=.019)., Conclusions: The results suggested an important role for GRK5 in the regulatory mechanisms of methadone dose and course of heroin dependence.

Majority of the heroin-dependent patients smoke cigarettes. Although it has been reported that the OPRM1 genetic polymorphism is associated with the brain mu-opioid receptor binding potential in cigarette smokers, there is no direct evidence showing the impact of plasma cotinine, a nicotine metabolite, on treatment responses to methadone maintenance treatment (MMT). In this study, we aimed to test the hypothesis that the genetic polymorphisms in the OPRM1 are associated with the methadone treatment responses and the severity of cigarette smoking directly measured by the plasma concentration of cotinine in a Taiwanese MMT cohort. Fifteen OPRM1 single-nucleotide polymorphisms (SNPs) were selected and genotyped on DNA samples of 366 MMT patients. Plasma concentrations of cotinine were measured by cotinine enzyme-linked immunosorbent assay. The plasma cotinine concentration had positive correlation with concentrations of methadone (P = 0.042) and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (P = 0.037). Methadone treatment non-responders, defined by a positive urine morphine test, had a higher plasma concentration of cotinine (P = 0.005), but a lower plasma concentration-to-dose ratio of both R- and S-methadone (P = 0.001 and 0.012, respectively) than the responders. OPRM1 genetic variants, rs1074287, rs6912029, rs1799971, rs12209447, rs510769, rs3798676, rs553202, rs7748401, rs495491, rs10457090, rs589046, rs3778152 and rs563649, were significantly associated with the plasma concentration of cotinine when using recessive model for genotypes (general linear model (GLM), P<0.038; false discovery rate (FDR)<0.035) and additive model for allele types (GLM, P<0.03; FDR<0.049) in association analyses. The G allele carriers of SNP rs1799971 (A118G) on exon 1 of OPRM1 gene had a lower plasma cotinine concentration than the A allele carriers (GLM, P = 0.029). OPRM1 genetic polymorphisms are associated with the plasma concentration of cotinine in a Taiwanese MMT cohort. Carriers with the major allele of SNP rs1799971 had a higher plasma cotinine concentration.