A single-cell transcriptome atlas of human euploid and aneuploid blastocysts.

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-ß and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development.

Transcriptomic profiles reveal the characteristics of oocytes and cumulus cells at GV, MI, and MII in follicles before ovulation.

BACKGROUND: The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the surrounding CCs. However, oocyte evaluation is primarily based on morphological parameters currently, which offer limited insight into the quality and competence of the oocyte. Here, we conducted transcriptomic profiling of oocytes and their CCs from 47 patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). We aimed to investigate the molecular events occurring between oocytes and CCs at different stages of oocyte maturation (germinal vesicle [GV], metaphase I [MI], and metaphase II [MII]). Our goal is to provide new insights into in vitro oocyte maturation (IVM). RESULTS: Our findings indicate that oocyte maturation is a complex and dynamic process and that MI oocytes can be further classified into two distinct subtypes: GV-like-MI oocytes and MII-like-MI oocytes. Human oocytes and cumulus cells at three different stages of maturation were analyzed using RNA-seq, which revealed unique transcriptional machinery, stage-specific genes and pathways, and transcription factor networks that displayed developmental stage-specific expression patterns. We have also identified that both lipid and cholesterol metabolism in cumulus cells is active during the late stage of oocyte maturation. Lipids may serve as a more efficient energy source for oocytes and even embryogenesis. CONCLUSIONS: Overall, our study provides a relatively comprehensive overview of the transcriptional characteristics and potential interactions between human oocytes and cumulus cells at various stages of maturation before ovulation. This study may offer novel perspectives on IVM and provide a reliable reference data set for understanding the transcriptional regulation of follicular maturation.

Telomeres cooperate in zygotic genome activation by affecting DUX4/Dux transcription.

Zygotic genome activation (ZGA) is initiated once the genome chromatin state is organized in the newly formed zygote. Telomeres are specialized chromatin structures at the ends of chromosomes and are reset during early embryogenesis, while the details and significance of telomere changes in preimplantation embryos remain unclear. We demonstrated that the telomere length was shortened in the minor ZGA stage and significantly elongated in the major ZGA stage of human and mouse embryos. Expression of the ZGA pioneer factor DUX4/Dux was negatively correlated with the telomere length. ATAC sequencing data revealed that the chromatin accessibility peaks on the DUX4 promoter region (i.e., the subtelomere of chromosome 4q) were transiently augmented in human minor ZGA. Reduction of telomeric heterochromatin H3K9me3 in the telomeric region also synergistically activated DUX4 expression with p53 in human embryonic stem cells. We propose herein that telomeres regulate the expression of DUX4/Dux through chromatin remodeling and are thereby involved in ZGA.

IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect.

Total fertilization failure (TFF) is an important cause of infertility; however, the genetic basis of TFF caused by male factors remains to be clarified. In this study, whole-exome sequencing was firstly used to screen for genetic causes of TFF after intracytoplasmic sperm injection (ICSI), and homozygous variants in the novel gene IQ motif-containing N (IQCN) were identified in two affected individuals with abnormal acrosome structures. Then, Iqcn-knockout mice were generated by CRISPR-Cas9 technology and showed that the knockout male mice resembled the human phenotypes. Additionally, we found that IQCN regulates microtubule nucleation during manchette assembly via calmodulin and related calmodulin-binding proteins, which resulted in head deformity with aberrant oocyte activation factor PLCζ. Fortunately, ICSI with assisted oocyte activation can overcome IQCN-associate TFF and male infertility. Thus, our study firstly identified the function of IQCN, highlights the relationship between the manchette assembly and fertilization, and provides a genetic marker and a therapeutic option for male-source TFF.

Pathogenic variant in ACTL7A causes severe teratozoospermia characterized by bubble-shaped acrosomes and male infertility.

Teratozoospermia is a common factor associated with male infertility. However, teratozoospermia characterized by bubble-shaped acrosomes (BSAs) has not yet been identified in men and the causative genes are unknown. The present study is of a patient with severe teratozoospermia characterized by BSA and carrying a variant (c.1204G>A, p.Gly402Ser) of actin-like 7A (ACTL7A). For further verification, we generated an Actl7a-mutated mouse model (p.Gly407Ser) carrying an equivalent variant to that in the patient. We found that homozygous Actl7a-mutated (Actl7aMut/Mut) male mice were sterile, and all their sperm showed acrosomal abnormalities. We detected by transmission electron microscopy that during acrosomal biogenesis, the acrosome detaches from the nuclear membrane in Actl7aMut/Mut mice. Furthermore, mutant ACTL7A failed to attach to the acroplaxome and was discharged by cytoplasmic droplets, which led to the absence of ACTL7A in epididymal spermatozoa in mice. The mutant sperm failed to activate the oocyte, and sperm-borne oocyte activation factor phospholipase C zeta (PLCζ) discharge accompanied by ACTL7A was observed, leading to total fertilization failure (TFF). Immunoprecipitation followed by liquid chromatography-mass spectrometry showed that several differentially expressed proteins participate in acrosome assembly and actin filament organization. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF in the couple with an ACTL7A pathogenic variant. Our study defined a novel phenotype of an acrosomal abnormality characterized by BSA, revealed the underlying mechanism of a pathogenic variant in ACTL7A and provided a genetic marker and potential therapeutic option for male infertility.

Biallelic variants in ZFP36L2 cause female infertility characterised by recurrent preimplantation embryo arrest.

BACKGROUND: Recurrent preimplantation embryo developmental arrest (RPEA) is the most common cause of assisted reproductive technology treatment failure associated with identified genetic abnormalities. Variants in known maternal genes can only account for 20%-30% of these cases. The underlying genetic causes for the other affected individuals remain unknown. METHODS: Whole exome sequencing was performed for 100 independent infertile females that experienced RPEA. Functional characterisations of the identified candidate disease-causative variants were validated by Sanger sequencing, bioinformatics and in vitro functional analyses, and single-cell RNA sequencing of zygotes. RESULTS: Biallelic variants in ZFP36L2 were associated with RPEA and the recurrent variant (p.Ser308_Ser310del) prevented maternal mRNA decay in zygotes and HeLa cells. CONCLUSION: These findings emphasise the relevance of the relationship between maternal mRNA decay and human preimplantation embryo development and highlight a novel gene potentially responsible for RPEA, which may facilitate genetic diagnoses.

Electrochemical Fingerprint Biosensor for Natural Indigo Dye Yielding Plants Analysis.

Indigo is a plant dye that has been used as an important dye by various ancient civilizations throughout history. Today, due to environmental and health concerns, plant indigo is re-entering the market. Strobilanthes cusia (Nees) Kuntze is the most widely used species in China for indigo preparation. However, other species under Strobilanthes have a similar feature. In this work, 12 Strobilanthes spp. were analyzed using electrochemical fingerprinting technology. Depending on their electrochemically active molecules, they can be quickly identified by fingerprinting. In addition, the fingerprint obtained under different conditions can be used to produce scattered patter and heatmap. These patterns make plant identification more convenient. Since the electrochemically active components in plants reflect the differences at the gene level to some extent, the obtained electrochemical fingerprints are further used for the discussion of phylogenetics.

Homozygous pathogenic variants in ACTL9 cause fertilization failure and male infertility in humans and mice.

Total fertilization failure (TFF) can occur during in vitro fertilization (IVF) treatments, even following intracytoplasmic sperm injection (ICSI). Various male or female factors could contribute to TFF. Increasing evidence suggested that genetic variations in PLCZ1, which encodes 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta-1 (PLCζ), is involved in oocyte activation and is a key male factor in TFF. In the present study, we explored the genetic variants in male individuals that led to TFF. A total of 54 couples with TFF or poor fertilization (fertilization rate < 20%) were screened, and 21 couples were determined to have a male infertility factor by the mouse oocyte activation test. Whole-exome sequencing of these 21 male individuals identified three homozygous pathogenic variants in ACTL9 (actin like 9) in three individuals. ACTL9 variations led to abnormal ultrastructure of the perinuclear theca (PT), and PLCζ was absent in the head and present in the neck of the mutant sperm, which contributed to failed normal calcium oscillations in oocytes and subsequent TFF. The key roles of ACTL9 in the PT structure and TFF after ICSI were further confirmed in an Actl9-mutated mouse model. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF and achieved live births in a couple with an ACTL9 variant. These findings identified the role of ACTL9 in the PT structure and the correct localization of PLCζ. The results also provide a genetic marker and a therapeutic option for individuals who have undergone ICSI without successful fertilization.

Traumatic carotid-cavernous fistula with perimedullary venous drainage and delayed myelopathy: A case report.

Traumatic carotid-cavernous fistula (TCCF) with perimedullary venous drainage and delayed myelopathy is a relatively rare clinical lesion. Endovascular embolization using embolic agents is the preferred treatment for patients with a poor collateral circulation. The present study describes the case of a 45-year-old male with TCCF, who presented with progressive cervical myelopathy for 1 month. A previous history of the patient included an anterior skull base fracture induced by a traffic accident 2 years prior. Cervical spinal magnetic resonance imaging (MRI) revealed dilated perimedullary veins and cervical spinal cord edema. Cerebral digital subtraction angiography revealed a direct CCF with perimedullary venous drainage. The patient received endovascular treatment with coils and an Onyx liquid embolic system to occlude the fistula, and his symptoms were relieved when he was discharged 3 weeks later. The patient then felt normal and a cervical spinal MRI revealed the disappearance of the perimedullary veins dilation and spinal cord edema at the 6-month follow-up. To the best of our knowledge, only three cases of CCFs with perimedullary venous drainage presenting with myelopathy have been previously reported. The present study also discussed the possible pathological mechanisms for this rare presentation. Moreover, it is suggested that the possibility of CCFs as a cause of cervical myelopathy needs to be taken into consideration.

Electrochemical Profile Recording for Pueraria Variety Identification.

The rapid identification of plant variety is valuable in both academic studies and crop production. However, rapid and accurate identification has been difficult because many varieties have very similar morphological characteristics and are susceptible to the effects of the growing environment. In this work, we established an electrochemical method for recording the electro-active profile of compounds in plant tissue. Because the chemical composition of different varieties is largely controlled by their genes, rather than a growing environment, this method has considerable potential for variety identification. Three varieties of Pueraria with sixteen locations were collected for confirming the feasibility of the proposed methodology. Principal component analysis and peak ratio analysis have been used for grouping the sample data. The results indicate the electrochemical profiles of three varieties can be distinguished using their voltammetric data.

CDCA8 regulates meiotic spindle assembly and chromosome segregation during human oocyte meiosis.

As a member of the chromosomal passenger complex, CDCA8 (cell division cycle associated 8) plays an important role in human mitosis, but its roles in human meiosis are unknown. Here, we show that CDCA8 expression is increased and its encoded protein has dynamic localization in human oocytes from germinal vesicle breakdown (GVBD) to metaphase Ⅱ (MⅡ), and that there are multipolar spindles, disordered chromosomes, and that microtubule assembly is affected after CDCA8 RNA interference (RNAi) in GV-stage oocytes. The GVBD and polar body extrusion (PBE) rates were not affected following CDCA8 depletion, but the PBE time was extended. There was no statistical difference between CDCA8 expression of oocytes from older and younger women, but the first polar body from older women was prone to chromosome abnormalities, and oocytes with such abnormalities had lower CDCA8 expression than oocytes with normal polar bodies. These results indicate that CDCA8 is associated with bipolar spindle formation, chromosome segregation, PBE during human oocyte meiosis, and that it may affect the incidence of aneuploidy embryos in older women.

New biallelic mutations in WEE2: expanding the spectrum of mutations that cause fertilization failure or poor fertilization.

OBJECTIVE: To investigate the genetic cause of fertilization failure or poor fertilization. DESIGN: Genetic analysis. SETTING: University-affiliated center. PATIENT(S): Twenty-four Chinese women who underwent assisted reproductive technology (ART) and had repeated fertilization failure or poor fertilization. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Twenty-four affected patients were subjected to whole-exome sequencing and candidate mutations were validated by Sanger sequencing. Single-cell reverse transcription was used to analyze the functional characterization of the splice-site mutation in vivo. Evolutionary conservation and molecular modeling analyses were used to predict the impact of missense mutations on secondary protein structure. Immunofluorescence was used to analyze the protein levels of WEE2 and phosphorylated CDC2. RESULT(S): Biallelic mutations in WEE2 were identified in 5 of 24 (20.8%) Chinese patients with fertilization failure or poor fertilization. Among these individuals we found a novel splice-site mutation, two novel missense mutations, and a previously reported frame-shift mutation. Splicing mutation c.1136-2A>G of WEE2 caused an alteration of the reading frame and introduced a premature stop codon (p.Gly379Glufs*6/p.Asp380Leufs*39). The missense mutations c.585G>C (p.Lys195Asn) and c.1228C>T (p.Arg410Trp) produced obvious changes in secondary protein structures. Immunostaining indicated that mutated WEE2 resulted in the loss of phosphorylated CDC2. The phenotypes of women carrying WEE2 mutations exhibited slight variability, from total fertilization failure to poor fertilization. CONCLUSION(S): Novel mutations in the known causative gene WEE2 were identified in 5 of 24 women with fertilization failure or poor fertilization, indicating a high prevalence of WEE2 mutations in Chinese women experiencing fertilization failure or poor fertilization.

Clinical and neonatal outcomes of patients of different ages following transfer of thawed cleavage embryos and blastocysts cultured from thawed cleavage-stage embryos.

BACKGROUND: Frozen-thawed embryo transfer (FET) has become a routine procedure in assisted reproductive technology (ART). In FET, although blastocysts cultured from thawed cleavage-stage embryos are associated with better perinatal outcomes. it may increase cycle cancellation due to no suitable embryo to transfer. The overall clinical outcomes following transfer of thawed cleavage-stage FET and blastocysts cultured from thawed cleavage-stage embryos in young and advanced age patients remains unclear. Therefore, we aimed to identify the optimal FET strategy in young and advanced age women who undergo FET. METHODS: This retrospective study included 16,387 thaw cycles. We retrospectively analyzed data of couples who had completed the first FET cycle. Two FET strategies were studied: transfer of thawed cleavage-stage embryos (strategy A) or blastocysts cultured from thawed cleavage-stage embryos (strategy B). The clinical and neonatal outcomes of two FET strategies were compared in young (<35 years) and advanced (≥35 years) age women. RESULTS: In young women, the clinical outcomes per transfer cycle were better in strategy B than strategy A. While the clinical pregnancy (59.29%, 52.60%) and live birth rates (49.37%, 43.88%) per thaw cycle were significantly higher in strategy A than in B. In women of advanced age, the clinical outcomes per transfer cycle were still better in strategy B than in A, and the clinical pregnancy (36.44%, 39.66%) and live birth rates (25.70%, 30.00%) per thaw cycle were significantly higher in strategy B than in A. CONCLUSIONS: FET of blastocysts cultured from cleavage-stage embryos showed higher efficiency for per transfer cycle whether in younger or advanced age women. Whereas, when cycle cancellations due to no suitable embryo to transfer were considered, cleavage-stage FET was found to be more suitable for younger women, while FET of blastocysts cultured from cleavage-stage embryos was better suited for women of advanced age.

The clinical and neonatal outcomes after stimulation of immotile spermatozoa using SperMagic medium.

To evaluate the efficiency and safety of SperMagic medium on stimulating the immotile spermatozoa in testicular sperm extraction (TESE) and absolute asthenozoospermia, 96 patients with TESE and 106 patients with absolute asthenozoospermia were enrolled in this study. The motile spermatozoa were detected in 47 TESE patients and 68 absolute asthenozoospermia and these patients were assigned to control group. The immotile spermatozoa in 49 TESE patients and 34 absolute asthenozoospermia were stimulated with SperMagic medium. Patients were treated by standard intracytoplasmic sperm injection (ICSI). There were no significant differences in fertilisation, cleavage, implantation, pregnancy, live birth and neonatal outcomes. SperMagic medium does not increase incidence of adverse neonatal outcomes and is a reliable tool for selection of viable spermatozoa in ICSI.

Inner cell mass incarceration in 8-shaped blastocysts does not increase monozygotic twinning in preimplantation genetic diagnosis and screening patients.

BACKGROUND: The use of assisted reproductive technology (ART) has been reported to increase the incidence of monozygotic twinning (MZT) compared with the incidence following natural conception. It has been hypothesized that splitting of the inner cell mass (ICM) through a small zona hole may result in MZT. In this study, using a cohort of patients undergoing preimplantation genetic diagnosis/screening (PGD/PGS), we compared the clinical and neonatal outcomes of human 8-shaped blastocysts hatching with ICM incarceration with partially or fully hatched blastocysts, and attempted to verify whether this phenomenon increases the incidence of MZT pregnancy or negatively impact newborns. METHODS: This retrospective study included 2059 patients undergoing PGD/PGS between March 1, 2013, and December 31, 2015. Clinical and neonatal outcomes were only collected from patients who received a single blastocyst transfer after PGD/PGS (n = 992). A 25- to 30-µm hole was made in the zona of day 3 embryos by laser. The blastocysts were biopsied and vitrified on day 6. The biopsied trophectoderm (TE) cells were analyzed using different genetic methods. One tested blastocyst was thawed and transferred to each patient in the subsequent frozen embryo transfer cycle. All the biopsied blastocysts were divided into three types: 8-shaped with ICM incarceration (type I), partially hatched without ICM incarceration (type II), and fully hatched (type III). ICM/TE grading, clinical and neonatal outcomes were compared between the groups. RESULTS: The percentage of grade A ICMs in type I blastocysts (22.2%) was comparable to that in type III blastocysts (20.1%) but higher than that in type II blastocysts (4.5%). The percentage of grade A TEs in type I blastocysts (4.2%) was comparable to that in type II (3.6%) but lower than that in type III (13.5%). There were no significant differences in clinical pregnancy, MZT pregnancy, miscarriage, live birth, MZT births, and neonatal outcomes between the groups. CONCLUSIONS: Compared to partially and fully hatched blastocysts, 8-shaped blastocysts with ICM incarceration showed relatively higher ICM and lower TE grades. ICM incarceration in 8-shaped blastocysts does not increase the incidence of MZT and has no negative effects on newborns in PGD/PGS patients.

Correlating charge heterogeneity data generated by agarose gel isoelectric focusing and ion exchange chromatography methods.

An isoelectric focusing method (IEF) has been used to assess the charge heterogeneity profile of a monoclonal antibody during the early stages of product development. A more precise and sensitive ion exchange chromatography (IEC/CEX) method was developed and implemented as development progressed and was used concurrently with IEF for lot release and to monitor charge heterogeneity. Charge variants resolved by both methods (IEC and IEF) were purified and characterized. Tryptic peptide mapping and N- linked oligosaccharide profile analyses of the IEC and IEF fractions indicated a structural correlation between the charge variants separated by these two methods. The major sources of molecular heterogeneity were due to the variation in the sialyated carbohydrate structure and heavy chain C-terminal lysine truncation. By monitoring the rates of change in the charge heterogeneity profiles of the monoclonal antibody stored at elevated temperatures by the IEC and IEF methods, a positive correlation between the two methods was established. This approach enabled replacement of the IEF method with the more precise IEC method.

Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

Isolation, purification, and antioxidant activities of degraded polysaccharides from Enteromorpha prolifera.

In the present study, purification and characterization of enzymatic hydrolysates of polysaccharide from Enteromorpha prolifera (HPE) are described. HPE was sequentially purified by DEAE Cellulose-52 chromatography and Sephadex G-100 chromatography to afford three fractions, namely, PHPE1, PHPE2, and PHPE3. Molecular weights of these three fractions were measured to be 103, 45.4, and 9.8kDa, respectively, using high performance gel permeation chromatography (HPGPC). The three fractions were evaluated for their antioxidant activities by determining their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion radicals. PHPE2 was found to possess the strongest scavenging ability. GC-MS analysis indicates that PHPE2 is mainly composed of mannose, xylose, and glucose.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity.

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry.

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.