We evaluated the association between neoantigen load and expression levels of immune-related genes. Moreover, using primary tumour cells established from pleural fluid of a breast cancer patient with carcinomatous pleurisy, we induced cytotoxic T lymphocytes (CTLs) by coculturing neoantigen peptide-pulsed dendritic cells (DCs) with autologous peripheral lymphocytes. The functions of CTLs were examined by cytotoxicity and IFN-γ ELISpot assays. Neoantigen load ranged from 6 to 440  and was positively correlated to the total number of nsSNVs. Although no associations between neoantigen load and mRNA expression of T cell markers were observed, the coculture of neoantigen-pulsed DCs and lymphocytes successfully induced CTLs ex vivo. These results suggest that neoantigen analysis may have utility in developing strategies to elicit T cell responses.Neoantigens are tumour-specific antigens that arise from non-synonymous mutations in tumour cells. However, their effect on immune responses in the tumour microenvironment remains unclear in breast cancer. We performed whole exome and RNA sequencing of 31 fresh breast cancer tissues and neoantigen prediction from non-synonymous single nucleotide variants (nsSNVs) among exonic mutations. Neoantigen profiles were determined by predictive HLA binding affinity (IC However, many breast cancer patients still die, especially from drug resistance4. Therefore, the development of new therapeutic strategies for breast cancer is critical.Treatment of breast cancer has advanced significantly in recent decades because of the development of new drug therapies7. Although the response rate to ICIs is limited and ICIs have been associated with immune-related adverse events, the response to ICIs is sometimes durable9. Notably, the existence of tumour antigen-specific T lymphocytes is an important factor for ICIs to be effective11. Among tumour antigens, peptides that contain amino acid substitutions caused by somatic non-synonymous mutations in tumour cells, but not in non-tumour cells, are called neoantigens.Recently, immune checkpoint inhibitors (ICIs) have shown anti-tumour efficacy in many cancer typesCD8A expression12. Another study showed that the number of neoantigens has a positive correlation to cytolytic activity in breast cancer, which is based on transcription levels of GZMA and PRF113. Conversely, other studies have shown a negative effect of high neoantigen load on the immune response in breast cancer15. Therefore, the effect of neoantigen burden on the immune response in breast cancer remains controversial.A high number of neoantigens is expected to elicit a strong immune reaction in the tumour microenvironment. A recent study of multiple cancer types including breast cancer showed that increased numbers of neoantigens are associated with higher 17. A recent study has reported the remarkable effect of adaptive transfer of neoantigen-specific T cells in a hormone-positive metastatic breast cancer patient18. Another study has demonstrated that neoantigens induce anti-tumour immunity in xenograft models19. However, studies on the therapeutic application of neoantigens in breast cancer remain limited and further evidence is required.Current research has focused on developing treatments that target neoantigensIn the current study, we performed neoantigen profiling of fresh tumour tissues from 31 breast cancer patients and examined the correlation between neoantigen load and the immune environment in breast cancer. We also examined whether dendritic cells (DCs) pulsed with in silico-predicted neoantigen peptides induce tumour-specific T lymphocytes in an ex vivo model.p < 0.0001, Fig. TP53 (13 cases), PIK3CA (5 cases), and TTN (5 cases), and five identical paired nsSNVs  were identified . The clinical characteristics of the 31 breast cancer patients are shown in Table 50 < 500 nM ranged from 9 to 834 , which showed a linear correlation to the number of nsSNVs . Expression levels of GZMA, GZMB and PRF1, which encode T cell activation markers, also did not show significant correlations .To investigate the effect of neoantigen load on the immune environment in breast cancer, the expression of immune-related genes was analysed by RNA-seq using the same sample that had been subjected to neoantigen analysis. There was no significant association between mRNA expression levels of T cell markers p < 0.01, Fig. p < 0.05, Fig. Neoantigen profiles can be applied to therapeutic applications such as cancer vaccines. Therefore, we examined whether CTLs were induced by coculturing lymphocytes with autologous DCs pulsed with the predicted neoantigen peptides. First, we performed neoantigen analysis of primary tumour cells established from pleural fluid of a TNBC patient with carcinomatous pleurisy. We synthesized 10 mutant peptides from the neoantigen profiles, which showed high affinity for HLA-A02:06 and mRNA expression with a read count of ≥ 2 Table . We also.We conducted neoantigen profiling of 31 breast cancers using an in silico prediction pipeline and analysed the correlation of neoantigen load with immune-related gene expression. We also performed ex vivo CTL induction experiments using TNBC patient-derived primary tumour cells and autologous PBMCs along with the predicted neoantigen peptides. In this study, we obtained the following findings: (1) the neoantigen profiles were highly unique among individuals; (2) neoantigen load showed a linear correlation with the total number of nsSNVs; (3) neoantigen load was not correlated to immune-related gene expression; (4) DCs pulsed with synthetic peptides predicted from our neoantigen analysis successfully induced CTLs ex vivo21. Previous studies have shown that 8–11mer peptides with a high affinity for HLA-class I elicit CD8 + CTLs that are the most effective cells to eliminate cancer22. Here, we performed neoantigen profiling of 31 breast cancer specimens based on the binding affinity of possible 8–11mer peptides, which harboured substituted amino acids altered by nsSNVs, to HLA class I molecules and mRNA expression levels of mutated genes. We succeeded in detecting neoantigens in all cases, which indicated good feasibility of neoantigen analysis in breast cancer regardless of the specimen site. Among the 1976 nsSNVs detected in the 31 cases, five pairs of identical nsSNVs were observed. Of these five genes, only AKT1 in both BC1 and BC14 remained as neoantigen candidates. Although frequently mutated genes such as TP53, PIK3CA, and TTN were found, there was only one pair of identical nsSNVs in PIK3CA. Accordingly, among the 1976 nsSNVs, only a pair of AKT1 remained as an identical neoantigen candidate. This result is consistent with a previous report showing that the majority of neoantigens are specific to the individual23. Considering that most neoantigens are derived from nsSNVs, the set of neoantigens in each patient varies greatly. This result indicates that neoantigen profiling may be important in developing highly personalized therapies.In silico neoantigen prediction analysis has recently been widely performed for many cancers, which is considered to be a potentially useful tool for clinical parameters and immunotherapies50 < 500 nM to HLA molecules is widely used25. In this study, we used two criteria: binding affinity of IC50 < 500 nM and RNA-seq data with one or more read counts that contained the mutated position. Although the RNA level required to elicit a T cell reaction is unclear, a study has shown that a single peptide-MHC complex induces cytotoxic T cells, which indicates that a very low expression level may be sufficient for T cell activation26. Therefore, we consider that even only one read count of RNA-seq can be a marker for a neoantigen peptide to elicit a T cell response. In this study, we referred to the number of neoantigen peptides predicted by not only the binding affinity to HLA class I molecules, but also RNA expression as “neoantigen load”. Both neoantigen load and the numbers of neoantigens predicted only by binding affinity to HLA class I molecules were positively correlated to the total number of nsSNVs. This is consistent with previous TCGA-based neoantigen analyses of breast cancers24. This result suggests that tumour mutation burden (TMB) simply reflects an increased opportunity of gaining neoantigens and that TMB can be a surrogate marker for neoantigen load. In the analysis by subgroup, we found that the neoantigen load was higher in TNBC cases than non-TNBC cases, which was consistent with previous reports24. Although the mechanism underlying the higher neoantigen load in TNBC compared with non-TNBC has not been elucidated, a possible cause is that, compared with luminal type breast cancer, which has specific tumorigenic factors such as signalling pathways via hormone receptors, TNBC requires a higher rate of mutations to gain tumour-driving mutations.Although there are no common criteria to predict neoantigens, a binding affinity of IC14. Another report also showed that a low TMB and neoantigen load were associated with high immune cell infiltration in TNBC15. Moreover, this trend has been reported in multiple tumour types27. One reason for the inability of a high neoantigen load to elicit immune-related gene expression may be the complexity of immunity in the tumour microenvironment, which includes multiple immune cell populations and cytokines29. Even if neoantigens are endogenously expressed and a immune response occurs, the existence of immune suppressive factors, such as regulatory T cells, myeloid derived suppressor cells, and immune suppressive cytokines, may counteract the immunogenicity of neoantigens. Another reason may be the neoantigen heterogeneity in breast cancers31. Several studies have demonstrated that tumour heterogeneity correlates with a reduced immune response32. Collectively, although neoantigens themselves induce immune responses, many other factors in the tumour microenvironment may cancel the immunogenicity of neoantigens.In contrast to our expectation, neoantigen load did not show positive correlations with mRNA expression of T cell markers or activated T cell markers. Consistent with our results, another report revealed an inverse association between immune metagene expression and TMB or neoantigen load in TNBC and the HER2-positive type33. However, in about half of the cases in the current study, we were unable to evaluate TILs because of the limited biopsy specimens that were available and prioritized them for neoantigen analysis. TIL evaluation of limited samples suggested no significant correlation between TILs and neoantigen load (data not shown). Consistent with this result, a recent study has shown no correlation between the number of nsSNVs and stromal TILs within each subtype of breast cancer34. We believe that the presence of neoantigen-specific TILs is more important than the number of TILs or neoantigen load and further analyses are required to address this issue.The relationship between neoantigen load and tumour-infiltrating lymphocytes (TILs) is a very interesting research area17. Using autologous tumour cells, monocyte-derived DCs, peripheral lymphocytes, and synthetic neoantigen peptides, we were able to successfully induce CTLs ex vivo. Importantly, CTLs releasing IFN-γ against autologous tumour cells indicated that the neoepitope predicted by our pipeline was expressed on the surface of tumour cells in the context of HLA class I molecules. If the neoantigen elicits effective anti-tumour immune responses in the tumour microenvironment, the tumour should not grow in patients. As we stated above, because of several immune suppressive factors in the tumour microenvironment, a neoantigen may not be able to elicit an immune response. Eliciting an immune response against a tumour-specific neoantigen ex vivo, where we can eliminate immune suppressive factors by adoptive neoantigen-specific CTL therapy as an example, may be a useful strategy to enforce a neoantigen-specific response. Although we recognize the limitations of a single preclinical model, these results demonstrated the theoretical rationale for clinical application of neoantigen peptides.In addition to the use of neoantigen profiles as a clinical immune parameter, there is great interest in their therapeutic use, such as for neoantigen vaccines and adaptive immunotherapy of neoantigen-specific CTLs36 and neoantigens with high affinity for HLA class II are reported to have an important role in the anti-tumour response38, these factors should be included in predicting neoantigens in future studies. Second, the proportion of tumour cells in the specimen probably affected the neoantigen load because a mutation was called for an mRNA read count relative to that of normal sequences. In the future, a neoantigen analysis system should be developed, which takes into account the tumour purity of specimens. Third, the number of specimens was small. Because breast cancers are reported to consist of multiple cancer subtypes39, a study should ideally be performed based on subtypes and a large number of specimens are needed for such analyses. Finally, we did not check whether the predicted neoantigen peptides were expressed on the tumour surface with MHC class I molecules and elicited specific CTLs in the tumour microenvironment. In pursuing the therapeutic application of neoantigen peptides, this may be an important direction to explore.Several limitations in this study should be stated. First, we predicted neoantigens only for each nonsynonymous SNV depending on the binding affinity for HLA class I. Because INDELs including gene fusions are reported to be good resources of highly immunogenic neoantigensWe performed neoantigen analysis of fresh tumour specimens from breast cancer patients. The neoantigen load was not positively correlated to immune-related gene expression. However, synthesized neoantigen peptide-pulsed DCs induced CTLs from peripheral lymphocytes ex vivo. Collectively, these results indicate that endogenous neoantigens in the tumour microenvironment of breast cancer may be insufficient to elicit an immune response in the tumour microenvironment. Therefore, inducing neoantigen-specific immune responses outside the tumour microenvironment by adoptive neoantigen-specific CTLs or neoantigen-pulsed DC vaccines may be alternative strategies to use neoantigen prediction analysis.All methods in this study were approved by the Ethical Committees of Kyushu University Hospital (No. 128) and Fukuoka General Cancer Clinic (FGCC-EC001) based on the Act on Securement of Safety on Regenerative Medicine in Japan. This study complied with the Declaration of Helsinki and written informed consent was obtained from all patients.This study included 31 patients with histologically confirmed breast cancer from Kyushu University and Fukuoka General Cancer Clinic. Fresh breast cancer specimens were obtained immediately after biopsies or tumour resection and placed in microtubes that contained the RNA stabilization solution RNAlater . PBMCs were obtained by leukapheresis procedure using Haemonetics CCS  in accordance with the manufacturer’s instructions. The leukapheresis product was diluted with RPMI-1640 medium  for density gradient isolation on Ficoll-Hypaque. After isolation, the cells were washed three times with RPMI-1640 medium and the obtained PBMCs were cryopreserved at − 80 °C. Tumour specimens and PBMCs were obtained at various time points from patients after diagnosis . After formation of colonies, they were cultured in DMEM/F12 supplemented with 10% fetal bovine serum  and cryopreserved at − 80 °C. We have previously reported the phenotype of the established primary breast cancer cells (MUK-BC1)41 and FPKM (fragments per kilobase of transcript per million mapped reads) values were calculated for immune-related gene expression analysis.Genomic DNA and total RNA were extracted from fresh tumours in RNAlater using the AllPrep DNA/RNA mini kit  in accordance with the manufacturer’s instructions. Control genomic DNA was extracted from patient-matched PBMCs. Whole exome libraries were prepared from 200 to 3000 ng genomic DNA using the SureSelect Human All Exon V6 kit  in accordance with the manufacturer’s instructions. RNA-seq libraries were prepared using the TruSeq Stranded mRNA Library Prep kit . The prepared whole exome and RNA-seq libraries were sequenced by a HiSeq 4000 at 2 × 100 bp or NovaSeq 6000 sequencer at 2 × 150 bp (Illumina). To analyse RNA-seq data, we used Cufflinks software42. For whole exome sequencing, the sequence reads were mapped to the human reference genome GRCh37/hg19 using Burrows–Wheeler Aligner (v0.7.10)43. Possible PCR duplicates, read pairs with a mapping quality of < 30, and mismatches of more than 5% were excluded. For RNA-seq, the sequence reads were mapped to the human reference genome GRCh37/hg19 using STAR (v2.4.0a)44. Somatic variants were called using Fisher’s exact test-based methods in accordance with the following parameters as described previously42: (1) base quality of ≥ 15; (2) sequence depth of ≥ 10; (3) variant depth of ≥ 4; (4) variant frequency in tumour of ≥ 10%; (5) variant frequency in normal samples of < 2%; (6) Fisher P-value of < 0.05. SNVs, indels, and splicing region were annotated using ANNOVAR45. We classified the somatic mutations as follows: (1) non-synonymous SNVs (nsSNVs); (2) splicing; (3) stop gain; (4) stop loss; (5) insertion/deletion (INDEL); (6) synonymous SNVs (sSNVs). Splicing mutation indicates that a single nucleotide variation occurred at the position of the splicing acceptor of the donor site.Sequence alignment and mutation calling were performed as described previously46. This strategy was used, as if the FPKM value was used, the mRNA expression level would be calculated from the sum of mutated and non-mutated genes. The total RNA-seq read counts were > 20,000,000 per specimen.We next used RNA-seq data of tumours to examine gene expression. The actual counts of sequence reads that covered the somatic mutated position were used to confirm mRNA expression of mutated genes47. We used only nsSNVs derived from possible peptides to predict HLA genotype-restricted neoantigens because the number of INDELs and fusions, if detected by RNA-seq, was much lower than that of nsSNVs and our neoantigen prediction algorithm for INDELs/fusions was under development and not yet validated42. Briefly, we examined all possible 8–11-mer peptides that harboured each substituted amino acid by filtering with the predicted binding affinity to HLA-A, -B, and -C of IC50 < 500 nM using NetMHCv3.4 and NetMHCpanv2.8 software49.On the basis of whole exome sequencing data from normal DNA of PBMCs, HLA class I genotypes  were determined by the OptiType algorithmIn the selection of neoantigen epitopes, we also considered the mRNA expression level of mutated genes by counting RNA-seq reads that contained the nsSNVs.DCs were generated from cryopreserved PBMCs. PBMCs were thawed and cultured in 6-well plates (BD FALCON) that contained RPMI-1640 medium for 30 min. After removal of floating cells and washing the plate with RPMI-1640 medium, adherent cells were cultured in complete DC medium that consisted of RPMI-1640 medium with recombinant human granulocyte/monocyte colony-stimulating factor  and recombinant human interleukin-4 . On day 6, the cells were stimulated with a maturation cytokine cocktail that contained human recombinant tumour necrosis factor-α  and interferon-α  for 18 h. Morphological changes were monitored by light microscopy and cells were examined by flow cytometric analysis. DCs that expressed high levels of HLA class I, HLA-DR, CD40, and a costimulatory molecule (CD86) with negative expression of CD14 (data not shown) were used for further experiments and referred to as mature DCs (mDCs).After pulsing mDCs with the indicated synthesized neoantigen peptides  in accordance with the manufacturer’s instructions. Briefly, 96-well plates with nitrocellulose membranes  precoated with a primary anti-IFN-γ antibody were pretreated with RPMI-1640 medium that containrf 10% autologous serum at 4 °C overnight. A total of 1 × 104 autologous immature DCs were added to each well along with the DC maturation cocktail and the cells were incubated overnight. Synthesized neoantigen peptide  was added to each well and the cells were incubated for 4 h. After three washes with RPMI-1640 medium, 1.0 × 105 or 1.5 × 105 autologous peripheral lymphocytes were added to each well and the cells were incubated for 48 h. After three washes with PBS, the secondary antibody was added to each well and the cells were incubated for 2 h. The plates were incubated with HRP reagent and stained with TNB (MABTECH). Spots were captured and analysed by an automated ELISpot reader 08 classic  and positivity of the neoantigen-specific T cell response was quantitatively defined as specific spots.The ELISpot assay was performed using the Human IFN-γ ELISpot3 per well) were seeded in ELISpot 96-well plates prepared as described above and cultured overnight. CTLs were then added to the wells (5 × 104 per well) and the cells were incubated for 4 h at 37 °C, followed by the same procedure to detect IFN-γ spots as described above.We also performed an ELISpot assay to examine the IFNγ-releasing ability of CTLs against autologous tumour. Briefly, target tumour cells (5 × 103 per well) were seeded in a 96-well flat-bottom plate and cultured overnight. The cells were then incubated at 37 °C for 1 h in the presence of calcein-AM. CTLs were then added to the wells (5 × 104 per well) and the cells were incubated with tumour cells for 4 h at 37 °C. Images were captured under a conventional fluorescence microscope  equipped with a colour CCD camera  and objective lens . All procedures were performed at 20–25 °C. Images were analysed using Lumina Vision software. The cytotoxicity percentage was calculated as (control fluorescence—sample fluorescence)/control × 100%.Target tumour cells (5 × 10t-test. Pearson correlation (r) was used for correlation analysis between all examined parameters. A p-value of < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism Version 8.3.0 .All data are expressed as the mean ± standard error of the mean (SEM). Differences between groups were assessed for statistical significance using the paired Student’s This study was approved by the Ethics Committees of Kyushu University Hospital (No. 128) and Fukuoka General Cancer Clinic (FGCC-EC001).Supplementary Information.


P = 0.0014, P = 0.0014, P = 0.0003, P = 0.0014, respectively). Moreover, the homocysteine level increased in the amniotic fluid of affected foetuses with MMACHC gene mutations. Of the four OTCD cases, genetic testing confirmed that two foetuses were affected and two were unaffected. However, the characteristic metabolite levels were within the reference range for all foetuses, including citrulline, orotic acid, and uracil. The genetic testing results were confirmed to be correct through the abortion tissue of the foetus and the postnatal follow-up. Our results suggest that mass spectrometry approaches are convenient method for improving the prenatal diagnosis of MMA. The characteristic metabolites C3, C3/C2, methylmalonic acid, and methylcitrate levels in amniotic fluid were reliable biochemical markers for the prenatal diagnosis of MMA.Methylmalonic acidaemia (MMA) and ornithine transcarbamylase deficiency (OTCD) are both intoxication-type inborn errors of metabolism (IEM). Presently, genetic testing is the primary method for prenatally diagnosing these diseases. However, some reports have demonstrated that mass spectrometry approaches can prenatally diagnose some forms of inborn errors of metabolism using amniotic fluid. Therefore, in this study, genetic and mass spectrometry approaches were used for prenatally diagnosing MMA and OTCD. We collected amniotic fluid samples from 19 foetuses referred, 15 cases were referred for MMA and 4 for OTCD. Of the 15 MMA cases, seven were affected, as determined by genetic testing and the metabolite levels; the characteristic metabolites propionylcarnitine (C3), C3/acetylcarnitine (C2) ratio, methylmalonic acid and methylcitrate levels were significantly higher than the reference range. Eight foetuses were unaffected, and the C3, C3/C2 ratio, methylmalonic acid and methylcitrate levels were within the reference range. The C3, C3/C2, methylmalonic acid, and methylcitrate levels in the amniotic fluid significantly differed between the affected and unaffected foetuses ( Methylmalonic acidaemia (MMA) and ornithine transcarbamylase deficiency (OTCD) are both common intoxication-type inborn errors of metabolism (IEM) in China , 2. The Presently, tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC/MS) are important methods for screening and clinically diagnosing IEM , 9. SomeSince the MS/MS and GC/MS methodologies are mature for diagnosing IEMs, this study describes our experiences with prenatally diagnosing IEM by measuring metabolites in the supernatant of amniotic fluid together with direct mutation analysis in MMA and OTC high-risk pregnancies.From January 2019 to June 2021, we recruited 15 unrelated families with MMA probands, 4 unrelated families with OTCD probands, and 73 pregnant women undergoing routine prenatal diagnoses with no history of IEM referrals. Five amniotic fluid samples from the referred MMA cases were collected by the Centre for Prenatal Diagnosis of Xinhua Hospital, Shanghai, China, all other samples were collected by our hospital . The probands were diagnosed based on clinical symptoms, GC/MS and MS/MS biochemical results, and genetic testing. Written informed consent was obtained from all participants. The study protocols were approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-Sen University (ethics committee batch number 2019ZSLYEC-105).The pregnant women with MMA or OTCD probands underwent amniocentesis by an experienced obstetrician; 10 mL of amniotic fluid was obtained from each pregnant woman. The amniotic fluid was centrifuged at 3000 r/min. Then, the amniotic fluid cells were collected for DNA extraction, and the supernatant was collected for mass spectrometry detection in separate sterile test tubes and stored at −80°C until use. All samples were collected before beginning the analyses, and the tests for all samples were performed at the same time.MMACHC)gene. The characteristic metabolite detected by MS/MS in the peripheral blood of OTCD patients was citrulline, and the metabolites detected by GC/MS were orotic acid and uracil , the C3/acetylcarnitine (C2) ratio (C3/C2), methylmalonic acid, and methylcitrate . We also, the C3/d uracil .The diagnostic criteria for characteristic metabolites of MMA and OTCD are consistent between amniotic fluid and peripheral blood. We used the 95th and 5th percentile mass spectrometry results from the 73 cases of normal (i.e. control) amniotic fluid to establish the upper and lower limits of the normal reference interval, the reference range of the different metabolites were displayed in the results section. All samples were sent to the Inborn Errors of Metabolism laboratory of the Sixth Affiliated Hospital of Sun Yat-sen University for MS/MS and GC/MS testing.Several amino acid and carnitine, methylmalonic acid, methylcitrate, and total Hcy determination kits  were used for the analysis. MS/MS  was used to detect the metabolites in 3 μL of amniotic fluid.GC/MS  was used to evaluate orotic acid and uracil in 100 mL of amniotic fluid. The amniotic fluid samples were processed by adding internal standard, deproteinizing, vacuum drying, and trimethylsilyl derivatization (BSTFA/TMCS) before testing.The QIAamp DNA Blood Mini Kit  was used to extract DNA from the amniotic fluid exfoliated cells, and then, polymerase chain reaction amplification and Sanger sequencing were used to test the candidate variants. The reference gene sequences were obtained from National Centre for Biotechnology Information GenBank database. Short tandem repeat analyses were performed to exclude maternal blood contamination and perform linkage analysis.Foetuses were considered affected by MMA if the characteristic metabolites in the amniotic fluid were higher than the upper limit of the reference range and the genetic test results of the amniotic fluid cells were consistent with the genetic test results of the proband.Foetuses were considered affected by OTCD if the concentration of citrulline in the amniotic fluid was lower than the lower limit of the reference range and the concentrations of orotic acid and uracil were higher than the upper limit of the reference range. Further, the genetic test results of the amniotic fluid cells must be consistent with the genetic test results of the proband.th and 5th percentiles from the cumulative levels of the 73 normal foetuses as the upper and lower reference limits, respectively. Wilcoxon rank-sum tests with exact P-values were performed to compare the levels of the characteristic metabolites between affected and unaffected groups. P < 0.05 was considered statistically significant.SPSS version 21.0  was used for the analyses. The reference range for each analyte was determined by a nonparametric approach, identified the 95We included 19 pregnant women with MMA or OTCD probands aged between 27 and 36 years with gestational ages from 16–21 weeks. We also included 72 pregnant women undergoing routine prenatal diagnosis  aged between 25 and 38 years with gestational ages from 16–23 weeks.MMUT) mutations, 5 with MMACHC mutations, and 1 with metabolism of cobalamin associated B (i.e. MMAB) mutations. The OTCD probands all had the ornithine transcarbamylase (OTC) gene mutations.th and 5th percentiles. The following values were considered normal: C3 = 0.16–1.53 μmol/L, C3/C2 = 0.08–0.57 μmol/L, methylmalonic acid 0.97–1.81 μmol/L, and methylcitrate 0.01–0.79 μmol/L. Further, the specific product of cblC also has Hcy 2.40–6.95 μmol/L, citrulline (Cit) > 3.30 μmol/L, orotic acid (Orotate) 0.00 mmol/mol creatinine, and uracil (Uracil) 0.00 mmol/mol creatinine.We established a reference range for each analyte using the 95MMACHC gene mutations was significantly higher than the concentration of the normal range and other affected amniotic fluid samples.We found no maternal contamination in the amniotic fluid samples by short tandem repeat analysis. n = 5) or no mutation (n = 3), and the median levels of C3, C3/C2, methylmalonic acid, and methylcitrate in the amniotic fluid were 0.87 μmol/L(range: 0.48–1.79), 0.16 (range: 0.07–0.25), 4.17 μmol/L(range: 1.30–10.60), 1.29 μmol/L(range: 0.80–1.63), and 0.22 μmol/L(range: 0.02–0.70), respectively. The C3, C3/C2, methylmalonic acid, and methylcitrate levels in the 7 samples amniotic fluid were within normal ranges. The C3 and Hcy levels in F1 was slightly higher than the normal range, while the levels of the other three metabolites were in the normal range. The C3, C3/C2, methylmalonic acid, and methylcitrate levels did not overlap between the affected and unaffected foetuses , orotic acid, and uracil  levels were in the normal ranges for all samples. The amniotic fluid metabolite and genetic testing results were inconsistent. Thus, the prenatal diagnosis was based on the genetic test results. The abortion tissue of the foetus and the postnatal follow-up confirmed the genetic testing.Currently, the standard method for prenatally diagnosing IEM is the detection of genetic mutations in amniotic fluid cells or chorionic tissues, but the disease-causing genes and mutation sites of the proband must be confirmed to complete the prenatal diagnosis. The genetic testing process is also complicated and slow, usually taking 10–25 days. Further, studies have shown that for autosomal recessive diseases, the foetus has only one causative variant as same as the proband, and the genetic testing results of the foetus cannot give a clear conclusion, but mass spectrometry can detect specific metabolite levels in the amniotic fluid, helping the prenatal diagnostic of foetus , 17. ImpSome studies have shown that MS/MS detection of C3, methylmalonic acid, and methylcitrate concentrations in amniotic fluid can be used for prenatally diagnosing MMA , 12. In MMACHC gene, which encodes a protein involved in the processing and trafficking of intracellular cobalamin. This subtype is also associated with increased Hcy and methylmalonic acid plasma concentrations . The cblC subtype, the most common subtype, is caused by biallelic pathogenic variants in the trations , 19. In OTC gene in two samples, consistent with the proband, there was no mutation in the other two samples. The metabolite and genetic testing results were inconsistent, suggesting that metabolite analyses using amniotic fluid at 16–22 weeks of pregnancy is not suitable for prenatally diagnosing OTCD. This result has not been suggested in other studies, and more data are warranted to prove the conclusion.OTCD, also known as hyperammonaemia type II, is an X-linked recessive genetic disorder; therefore, almost all hemizygous males develop this disease. Approximately, 20% of female carriers present some neurocognitive disorders due to random X-inactivation. However, the symptoms are substantially milder than those experienced by male patients. Presently, genetic testing is the primary method for prenatal diagnosis of OTCD; although male foetuses can be diagnosed, due to the unpredictability of random X chromosome inactivation, it is impossible to determine whether foetal female carriers have the disease. Prenatal diagnosis of OTCD in female foetuses using mass spectrometry will therefore be difficult. In this study, four women were referred with OTCD, and all foetuses were confirmed to be male through genetic testing. The MS/MS and GC/MS results showed that the citrulline, orotic acid, and uracil levels in all amniotic fluid samples were within the reference range, suggesting that none of the foetuses had OTCD. However, the genetic testing indicated a c.512A > G hemizygous mutation and c.867 + 1G > C hemizygous mutation in the In summary, mass spectrometric approaches are a convenient prenatal diagnostic method for pregnancies at risk for MMA. Characteristic metabolite  levels in the amniotic fluid were reliable biochemical markers for prenatally diagnosing MMA. A combined genetic and biochemical approach would provide increased diagnostic accuracy.The studies involving human participants were reviewed and approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University (Ethics Committee Batch no. 2019ZSLYEC-105). The patients/participants provided their written informed consent to participate in this study.XX conceived and designed the study. CS wrote the manuscript draft. SL, ZD, HH, and XX enrolled the patients. YG performed amniocentesis. CS and SL performed the metabolite test, mutation test, and data analysis. All authors read and approved this manuscript.This work was supported by Guangzhou Science and Technology Plan Project (No. 201604020154) and Guangdong Science and Technology Plan Project (No. 2020A1414010111).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gyps fulvus). Griffons’ foraging activity increased in the pasturelands occupied by transhumant herds, which implied greater vulture abundance at the landscape level during the livestock season. In contrast, facultative scavengers were more abundant without transhumant livestock herds, and the abundance of wild ungulates did not change in relation to livestock presence. We conclude that fostering transhumance and other traditional farming systems, to the detriment of farming intensification, could favour vulture conservation.In recent decades, intensive techniques of livestock raising have flourished, which has largely replaced traditional farming practices such as transhumance. These changes may have affected scavengers’ behaviour and ecology, as extensive livestock is a key source of carrion. This study evaluates the spatial responses of avian scavengers to the seasonal movements of transhumant herds in south-eastern Spain. We surveyed the abundance of avian scavengers and ungulates, and analysed the factors affecting the space use by 30 GPS-tracked griffon vultures (The online version contains supplementary material available at 10.1007/s13280-021-01668-x. Gyps fulvus; Parra and Tellería Neophron percnopterus; Mateo-Tomás and Olea Humans and scavengers have maintained a close and changing relationship since our origins .However, the profound change in livestock products demand and management in the last few years could strongly influence scavenger behaviour and conservation. Both economic development and human population growth have led to an increasing demand in livestock products and a shift towards industrialization , an area that houses the largest number of transhumant sheep in western Europe. To address this, we firstly explored the relationship between the abundance of avian scavengers (both obligate and facultative) and the abundance of wild and domestic ungulates at the landscape level, as inferred by field surveys. We secondly analysed the individual spatial responses of GPS-tracked griffon vultures to the presence and absence of transhumant herds. We focussed on the griffon vulture because it is the most abundant vulture species in Europe , a mountain region in south-eastern Spain. The climate is Mediterranean, with annual rainfall ranging between 300 and 1600 mm, and a mean annual temperature of 12–16 ºC. The area is covered mainly by pine  and oak (Quercus ilex and Q. faginea) forests (Rivas-Martínez Gypaetus barbatus and Egyptian vultures) and one species that is occasionally present (cinereous vultures Aegypius monachus) , common ravens (Corvus corax), carrion crows (C. corone), Eurasian magpies (Pica pica) and Eurasian jays (Garrulus glandarius), and also mammals like red fox (Vulpes vulpes) and wild boar (Sus scrofa) , fallow deer (Dama dama), mouflon  and Iberian ibex (Capra pyrenaica) , but also goats (Capra aegagrus hircus), cattle (Bos taurus) and horses .In the highest part of this natural park, there is a plateau called “Campos de Hernán Perea”  and ungulates (both domestic and wild species) between the seasons with and without transhumant livestock. For birds, we conducted 75 systematic 30-min point counts , which were attached using a backpack harness. These devices were configured according to weather conditions (see Table S1). Birds were monitored between January 2015 and December 2018, unless they died or the device technically failed (Table S2). All the individuals were sexed by molecular procedures .We firstly used non-parametric Wilcoxon rank tests to explore changes in the abundance of avian scavengers and ungulates between seasons. The absence of normality in these variables was confirmed by the Shapiro–Wilk test , “breeding” status (breeding/non-breeding), “sex”  and “year” . The individual (“ind.”) was included as a random term. The process for model construction and selection, and for calculating the candidate models’ performance, was like that described above.https://cran.r-project.org/).All the analyses were done in R software, version 3.6.1 (R Core Team p < 0.05); Fig. Obligate scavengers were more abundant during the livestock season , which became lower after excluding the variable “sex”  abundance in the studied pastureland increased after transhumant herds had arrived. This agrees with previous research conducted in northern Spain, which found that the occurrence of griffon vultures increased at the roosting sites near transhumant sheep’s summer pastures (Olea and Mateo-Tomás 2) compared to the griffon vulture home range in southern Spain , which show different resource exploitation patterns Below is the link to the electronic supplementary material.


ALG13, characterized to date only in about 60 individuals . This is an important preliminary step in the understanding of the pathogenesis of the disease associated with this variant in the rare genetic condition. The disease is assumed to be a disorder of N-glycosylation given that this is the only known function of the ALG13 protein. Despite this, protein electrophoresis, which is abnormal in most conditions due to abnormalities in N-glycosylation, has been normal or only mildly abnormal in the ALG13 patients. Methods: Nuclear magnetic resonance (NMR) spectroscopy in conjunction with multivariate and univariate modelling were used to analyze the metabolic profile of the blood serum samples acquired from the studied patients. Results: Three metabolites were identified as potential biomarkers: betaine, N-acetyl-glycoprotein, and carnitine. Conclusions: Since presented data are the first to be collected so far, they need be verified in further studies. Our intention was to turn attention toward possible CDG-ALG13 laboratory markers that would have clinical significance.Background: ALG13-CDG belongs to the congenital disorders of glycosylation (CDG), which is an expanding group of multisystemic metabolic disorders caused by the N-linked, O-linked oligosaccharides, shared substrates, glycophosphatidylinositol (GPI) anchors, and dolichols pathways with high genetic heterogeneity. Thus, as far as clinical presentation, laboratory findings, and treatment are concerned, many questions are to be answered. Three individuals presented here may serve as a good example of clinical heterogeneity. This manuscript describes the first metabolomic analysis using NMR in three patients with epileptic encephalopathy due to the recurrent c.320A>G variant in  Glycosylation is the most common post-translational modification of proteins and lipids, and its biological role is crucial in the processes of development, growth, and functioning of the body. More than 140 different congenital disorders of glycosylation (CDG) types associated with abnormal protein glycosylation are currently described ,3,4,5,6.ALG13-CDG presents as an early infantile epileptic encephalopathy. According to the most recent data, the c.320A>G p.Asn107Ser) variant is the most frequent female predominant mutation 7Ser vari,12,13,14ALG13. The metabolome characteristics of these three female patients are presented, giving assumptions for further studies that could verify the role of three metabolites as biomarkers suitable for clinical or treatment assessment. As we believe, it is an important preliminary step in the understanding of the pathogenesis of the disease associated with this variant. It is also a very first approach to employ NMR techniques in CDG studies. This is an important preliminary step in the understanding of the pathogenesis of the disease associated with this variant, which has been seen in nearly 60 patients worldwide .This paper describes the first metabolomic analysis using proton high resolution nuclear magnetic resonance (1H HR NMR) spectroscopy in three patients with epileptic encephalopathy due to the recurrent c.320A>G variant in The study was approved by the ethical committee of the Silesian Medical University, and written informed consent was obtained from each child’s parent(s) or guardian for the participation of their child in the study. All methods were performed in accordance with the relevant guidelines and regulations.ALG13 gene are described . Patient 3 was also heterozygous for c.3433C>T  in the CACNA1A gene, which is of unknown significance; its potential impact on the clinical state is thus impossible to define. The age of the patients at the last visit ranged from 2 to 5 years 8 months.Three female probands with de novo c.320A>G variant in the Patient 1The patient was the second child of nonconsanguineous Polish parents born from an uneventful pregnancy at 38 weeks of gestation with a birth mass of 2430 g. Since birth, epileptic seizures were observed in the form of myoclonic seizures and facial grimaces. Valproic acid and vigabatrin treatment was used. At the age of 8 months of age, she presented with an axial hypotonia and psychomotor retardation. MRI of the brain was normal with myelination appropriate for the patient’s age. Hypsarrhythmia was observed in EEG pattern.At the last follow-up at 2 years of age, the child presented with microcephaly and developmental delay, but slow improvement was observed . A visual impairment was diagnosed in the form of astigmatism and hyperopia. No seizures were observed. The monotherapy with valproic acid was continued.Patient 2The patient was the second child of nonconsanguineous Polish parents born from an uneventful pregnancy at 41 weeks of gestation with a birth mass of 4510 g. Since the age of 4 months, epileptic seizures were observed, firstly as classical tonic-clonic seizures (treated with vigabatrin), then as myoclonic seizures . Since the age of 6 months, the aggravation of seizures was observed; thus, ACTH (adrenocorticotropic hormone) treatment was implemented. The child at that time presented with an axial hypotonia, a developmental delay, and microcephaly. Hypsarrhythmia was observed in the EEG pattern. MRI of the brain revealed slightly enlarged lateral ventricles with myelination appropriate for the patient’s age. There was no improvement in epileptic seizures. Topiramate was introduced and then switched to lamotrigine. Drug-resistant epilepsy was finally diagnosed.At the last follow-up at 4 years of age, the child presented with microcephaly and severe global developmental delay, but slow improvement in motor skills were observed . A visual impairment was diagnosed in the form of astigmatism and hyperopia. Seizures were observed every day or every second day.Patient 3The patient was the second child of nonconsanguineous Polish parents born from pregnancy complicated by gestational diabetes at 38 weeks of gestation with a birth mass of 3320 g. A spontaneous miscarriage of a first pregnancy at 8–10 weeks of gestation was observed. Since the age of 4 months, myoclonic seizures were observed. At 6 months of age, the child presented with opisthotonos, developmental delay, strabismus, and relative macrocephaly (OFC 90–97pc). Hypsarrhythmia was observed in the EEG pattern. MRI of the brain was normal with myelination appropriate for the patient’s age. In the next several months, the aggravation of seizures, myoclonic in form, was observed; thus, lamotrigine, and then vigabatrin, and finally ACTH treatment were implemented.At the last follow-up at 5 years and 8 months of age, the child presented with relative macrocephaly (OFC 90–97pc), a severe global developmental delay, but a slow improvement of the motor skills was observed . The seizures were observed every day or every second day. The current treatment includes valproic acid, topiramate, and levetiracetam.Nuclear magnetic resonance (NMR) spectroscopy in conjunction with multivariate and univariate modelling were used to analyze the metabolic profile of the blood serum samples acquired from the studied patients. The obtained metabolic profiles were then compared with the ones from the pediatric drug-resistant epilepsy patients (EG) as well as from the pediatric reference group (RG). The characteristics of the EG and RG groups and the detailed description of the blood samples collection, as well as the NMR acquisition protocol, are described in , and a bThe EG group consisted of 28 patients suffering from drug-resistant epilepsy . The types of seizures and epilepsy as well as the epilepsy syndromes were defined in accordance with the International League Against Epilepsy Classification and Terminology (ILAE 2017). All EG patients underwent diagnostic protocol including EEG, videoEEG, 24-hour EEG (in selected sapienti), laboratory tests for inborn errors of metabolism, chromosomal analysis, and molecular study. The RG group included 20 patients . All children were free of neurological symptoms, developmental delay, or chronic diseases. The routine laboratory tests, EEG, and MRI of the brain showed no abnormalities.The ALG13 group consisted of three patients (the patient characteristics are provided in The sera samples (500 µl) from the overnight peripheral blood were mixed with 500 µL of phosphate buffer (pH 7.4) with D2O and TSP, and the aliquots of 600 µL were transferred into 5 mm NMR tubes  and stored at 4 °C until the spectroscopic analysis. 1H NMR spectra were acquired on a Bruker 400 MHz Avance III spectrometer  at the temperature of 310 K and with a constant receiver gain (90.5). NOESY (nuclear Overhauser effect spectroscopy), CPMG (Carr–Purcell–Meiboom–Gill), DIFF (diffusion edited), and two-dimensional JRES (J-resolved) spectra were acquired for each blood serum sample. After automatic post-processing, the spectra were aligned to the alanine signal at 1.5 ppm and bucketed (with a bucket size of 0.02 ppm) over the region between 0.5 and 9.0 ppm with the exclusion of the water residual signal. No normalization was applied. The NMR-visible metabolites were quantified based on the 1D positive projections of the JRES spectra, and the peaks were integrated by area using AMIX . The 1D positive projections of the JRES spectra were used for quantification of low molecular weight metabolites, while the lipid signals were quantified based on the diffusion edited spectra. The peaks were integrated by area using AMIX , and the integrals were measured in the spectral regions defined individually for each low molecular weight metabolite, whereas in the case of the lipid signals, a 0.12 ppm range around a particular peak was applied. Sixty-eight NMR signals  were quantified. The multivariate analyses were performed on the bucketed NMR spectra as well as on all quantified metabolites.Scores plot (PCA and OPLS-DA)—used to observe the patterns or class separation in the data.y axis (p-corr) had high reliability, while the variables more shifted along the x axis showed a greater magnitude of the changes. The ideal candidate for a biomarker has both high reliability and magnitude.S-plot (OPLS-DA)—giving insight into the variables’ importance for the observed class separation. The variables shifted away from the plot origin along the The multivariate tools used in the present study were: unsupervised principal component analysis (PCA) and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA). The main results for the multivariate analyses are presented graphically in two types of plots:X) and the information about belonging to a given class (denoted in the form of a zero-one Y matrix). The orthogonality of the OPLS-DA method means that the separation between classes on the scores plot is always illustrated along the x axis, while the intra-group variability is presented along the y axis. This markedly facilitates the interpretation of the results. The amount of variation in the data that is correlated to class separation is given by a predictive component, while an orthogonal component gives the amount of uncorrelated variation between X and Y matrices. An extensive discussion of these multivariate methods and the plots presenting the results is available in [PCA visualizes the directions of the largest variation in the data. However, these directions may not always reflect the separation between individual classes. In such a situation, supervised methods are useful, in particular OPLS-DA. OPLS-DA models the relation between the data matrix  test.In addition to multivariate methods, a univariate Kruskal–Wallis ANOVA was used  to further investigate the statistical differences between the studied groups.The multivariate PCA, OPLS-DA models and the S-plots for the ALG13, EG, and RG groups are shown in The diagnostics of the discussed PCA and OPLS-DA models are provided in p-value > 0.999, p-value = 0.08, The multivariate modelling failed to distinguish the ALG13-CDG cases from the EG group—the resulting OPLS-DA model d,f showeBased on the results from OPLS-DA, we used the scatter plots and the univariate statistics in order to investigate these three metabolites  further. A comparison of the relative intensities of betaine, NAG, and carnitine signals between the ALG13, EG, and RG groups is shown in p-value = 0.014) and fell in the upper range of variation of the EG group . NAG, in turn, revealed a considerable internal difference in the ALG13 c.320A>G carrier cases  came closer to significance   [To date, ALG13-CDG has been reported in more than 60 patients, most of which results from c.320A>G variant  ,14.Regarding epilepsy, most known ALG13-CDG patients have been treated with a combination of many antiepileptic drugs (AEDs), which unfortunately did not lead to adequate seizures control ,18. ThusThe presented NMR-based metabolomics results obtained for patients with ALG13-CDG caused by a c.320A>G variant provide an interesting preliminary insight into the laboratory characteristics of one of the glycosylation disorders. The mammalian glycome consists of up to several thousand glycan structures and may be larger even than the proteome. Aberrations in the glycosylation anabolic and catabolic mechanisms may disturb cell adhesion, cell signaling, and endocytosis, with congenital disorders of glycosylation (CDGs) affecting the central and peripheral nervous system, the endocrine system, and coagulation ,16,19,20ALG13 c.320A>G carrier cases are restricted to three metabolites: betaine, NAG, and carnitine; however, only betaine was found to be an ALG13 specific marker statistically confirmed by the univariate tests . The other two compounds indicated by the OPLS-DA models, carnitine and N-acetyl-glycoprotein, can be only treated as the compounds of a possible importance; however, their role should be confirmed in larger group studies. While the ALG13 and RG groups were clearly separated in the OPLS-DA scores plot , presumably due to a small size of the ALG13 group. The separation between ALG13 and EG was markedly weaker . In this case, apart from the small ALG13 group size, the shared similarities of the metabolic profiles of both groups also seem to be of importance. This supposition is supported by the results obtained for a model that differentiates between the EG and RG groups for prediction of class membership of ALG13 patients (data not shown). The ALG13 samples included in this classification were identified by this model as EG (Patients 2 and 3) and as ambiguous (Patient 1). For such classification of Patient 1, the mildest spectrum of ALG13-CDG as compared with the other two cases may be responsible.In our study, the multivariate metabolomic profiling and the descriptive statistics of the NMR spectra of the blood sera revealed that the metabolic alterations presumably characteristic for the res plot c, the moy weaker d, as refBetaine, a small amino-acid, with its primary role to serve as a methyl group donor in liver metabolism, is also used as an osmoprotectant in most tissues in the body ,22. The ALG13 are claimed to be near-normal [Glycosylation produces different types of glycans (or glycoconjugates) that are typically attached to cellular proteins and lipids. Approximately half of all proteins typically expressed in a cell undergo glycosylation. There are mainly two categories of glycosylation: N-glycosylation and O-glycosylation. N-glycans are linked to the amide group of asparagine, while O-glycans are linked to the hydroxyl group of serine or threonine ,23. One r-normal ; thus, tThe third metabolite identified by OPLS-DA and with close to statistical significance difference between ALG13 and RG —carnitinThe main limitation of the study is the very small size of the ALG13 group. However, due to the extreme rarity of the disease, the three analyzed cases account for 5% of all diagnosed cases worldwide. Another limitation is related to OPLS-DA being prone to overfitting. Keeping this in mind, the results present the OPLS-DA model calculated on the quantified metabolites, which drastically reduced the number of the variables  and, at least to a small extent, reduced the risk of overfitting. Furthermore, the model was internally cross-validated as well as verified using univariate statistical methods.ALG13 variant, in understanding the complex pathophysiology underlying a glycosylation deficiency, and in establishing new therapeutic approaches.Betaine, N-acetyl-glycoprotein, and carnitine were indicated by OPLS-DA to be possible biomarkers of ALG13; however, only betaine was confirmed by the univariate ANOVA tests to be specific for ALG13-CDG. Hopefully, further studies performed on a larger ALG13 group could verify the role of these possible markers for classifying ALG13-CDG disease severity or/and monitoring disease treatment with anti-epileptic drugs. Defects in the metabolism of amino acids are claimed to lead to pathology of the nervous system and many other organs ,32. Meta


Varicella and herpes zoster are mild symptoms-associated diseases caused by varicella–zoster virus (VZV). They often cause severe complications (disseminated zoster), leading to death when diagnoses and treatment are delayed. However, most commercial VZV diagnostic tests have low sensitivity, and the most sensitive tests are unevenly available worldwide. Here, we developed and validated a highly sensitive VZV diagnostic kit based on the chemiluminescent immunoassay (CLIA) approach. VZV-glycoprotein E (gE) was used to develop a CLIA diagnostic approach for detecting VZV-specific IgA, IgG, and IgM. The kit was tested with 62 blood samples from 29 VZV-patients classified by standard ELISA into true-positive and equivocal groups and 453 blood samples from VZV-negative individuals. The diagnostic accuracy of the CLIA kit was evaluated by receiver-operating characteristic (ROC) analysis. The relationships of immunoglobulin-isotype levels between the two groups and with patient age ranges were analyzed. Overall, the developed CLIA-based diagnostic kit demonstrated the detection of VZV-specific immunoglobulin titers depending on sample dilution. From the ELISA-based true-positive patient samples, the diagnostic approach showed sensitivities of 95.2%, 95.2%, and 97.6% and specificities of 98.0%, 100%, and 98.9% for the detection of VZV-gE-specific IgA, IgG, and IgM, respectively. Combining IgM to IgG and IgA detection improved diagnostic accuracy. Comparative analyses on diagnosing patients with equivocal results displaying very low immunoglobulin titers revealed that the CLIA-based diagnostic approach is overall more sensitive than ELISA. In the presence of typical VZV symptoms, CLIA-based detection of high titer of IgM and low titer of IgA/IgG suggested the equivocal patients experienced primary VZV infection. Furthermore, while no difference in IgA/IgG level was found regarding patient age, IgM level was significantly higher in young adults. The CLIA approach-based detection kit for diagnosing VZV-gE-specific IgA, IgG, and IgM is simple, suitable for high-throughput routine analysis situations, and provides enhanced specificity compared to ELISA. Varicella–zoster virus (VZV), also known as human herpesvirus-3 (HHV-3), belongs to the α-herpesviridae subfamily . It is rAlthough considered among the mild-symptom diseases, VZV-related diseases are highly morbid. The most life-threatening complications include mental development deficit, meningoencephalitis, and post-infectious encephalopathy, in varicella cases. In herpes zoster cases, complications include vasculitis, zoster sine herpete, and post-herpetic neuralgia. A lack of early diagnosis results in treatment delays, which usually leads to fatalities, especially in newborns, elders, organ transplant recipients, and immunocompromised people experiencing disseminated herpes zoster ,5,6,7,8.Nowadays, VZV vaccination has led to a significant decrease in the incidence of varicella, particularly in countries where vaccination programs have been implemented and well followed ,10,11. CWhile the diagnosis of VZV infection is needed in both countries with well-established and non-implemented VZV-vaccine programs, routine biological diagnostics has become challenging, as many currently available diagnostic tests have low sensitivity/specificity ,13,23. TAlthough molecular diagnostics, including viral isolation from vesicular fluid cultures and swab samples, and nucleic acid detection (by PCR), are the most sensitive in VZV diagnostic ,28, antiSeveral studies support that the chemiluminescent immunoassay (CLIA) approach for diagnosing serum/plasma viral antigen-specific IgA, IgG, or IgM has better diagnostic performance among the known immunoassays, including manual ELISA ,32,33. MTherefore, we developed and validated a highly sensitive and specific diagnostic kit based on the CLIA approach for diagnosing VZV infections.The developed CLIA-based VZV diagnostic approach demonstrated improved diagnostic accuracy, as it could detect very low IgA, IgG, and IgM titer in patients at the early stage of the VZV infection. Moreover, as the diagnosis process is automated, time-saving, and suitable for high-throughput situations, it can be used for routine diagnoses of VZV infections.Overall, 29 people  with an A total of 62 blood samples from the 29 retained VZV-patients and 453 plasmas/sera from random healthy people (used as negative controls) were obtained to test the developed diagnostic approach.As aforementioned, three ELISA kits  were used to assess the VZV-specific IgA, IgG, and IgM, respectively, in VZV-patient samples from the 29 included VZV-patients. Sixteen samples from random healthy people were used as negative controls. According to the manufacturer’s instructions, these ELISA assays were considered standard for VZV-specific antibody detection in patients to confirm and stratify the retained patients as true-positive or equivocal groups. All samples were first serially diluted and assessed for VZV immunoglobulin detection. As a result, all patient samples (but not those from negative controls) reacted with VZV glycoproteins in a concentration-dependent manner . These rSpecifically, of the 29 included patients, 21 showed moderate to high reactivity, even at high dilution for IgA, IgG, and IgM detection, respectively. With OD450 ≥ 1 at 1/100 dilution, all these 21 patients were considered true-positive for IgA, IgG, and IgM according to the ELISAs’ manufacturer instructions .In contrast, eight patient samples  showed inconsistent results in IgA, IgG, and IgM detection, respectively. Precisely, each patient sample showed equivocal results (OD450 between 0.1 and 0.21 at 1/100 dilution) for at least one of the three antibody isotypes IgA, IgG, and IgM . TherefoThe highly purified VZV-gE antigen  was usedThen, to assess the performance of the CLIA approach, the cohort of 42 samples from the 21 true-positive patients and 453 independent samples (plasma or serum) from healthy people were tested by the developed VZV-gE-IgA/IgG/IgM kit. Before testing, samples were pre-treated (virus-inactivated) and diluted 20 times with dilution buffer (PBS) supplemented with 2% BSA. ROC analysis results showed sensitivities of 95.2% (IC95%: 76.2–99.9%), 95.2% (IC95%: 83.8–99.4%), and 97.6% (IC95%: 87.4–99.9%) and specificities of 98.0% (IC95%: 96.3–99.1%), 100% (IC95%: 99.2–100%), and 98.90% (IC95%: 97.4–99.6%) for IgA, IgG, and IgM detection, respectively . The cutIt is well known that IgMs are the first immunoglobulins produced at the first contact with viruses. Days after infection, IgM titer in the blood decreases while IgA and IgG titer increases . As prevTo assess the diagnostic ability of the CLIA kit to diagnose the samples classified as equivocal by a standard ELISA tests, individual and combined detection of IgA, IgG, and IgM were conducted with the CLIA-based diagnostic approach in the eight-patient cohort. These results were analyzed along with previous cohort results and compared to those from ELISA. The comparative analysis was based on previously obtained IgA, IgG, and IgM criteria  A–C and tp-value = 0.567)   A. Regard= 0.567) , Table 2< 0.001) B. RegardWe aimed to determine the diagnostic performance of the CLIA-based IgA, IgG, and IgM detection kit in diagnosing VZV infection in a random population. To this end, we considered the equivocal samples as positive and used the whole cohort, which consisted of the 62 independent samples from included patients and the 453 negative samples from healthy donors. The ROC analysis showed sensitivities of 74.2% (IC95%: 61.5–84.5%), 69.4% , and 93.6% (IC95%: 84.3–98.2%), and specificities of 96.0% (IC95%: 93.8–97.6%), 99.8% (IC95%: 98.0–100%), and 98.0% (IC95%: 96.3–99.1%) for the diagnostic of VZV-gE IgA, IgG, and IgM, respectively . InteresPatient clinical data along with CLIA-based diagnostic results were analyzed. As a result, IgM titer was higher in all patients, while IgA and IgG titers were lower in patients with equivocal results A. MoreovTo assess whether there is any significant difference between the IgA, IgG, and IgM levels regarding VZV-patient age, the patient cohort was divided into two groups (≤35 years old and >35 years old). CLIA-associated diagnostic results from the 28 patients with reported age were considered . AnalyseReliable assays in the immunological diagnosis of VZV with the best performance are essential in the era of vaccine program implementation, their effectiveness evaluation, and for VZV-associated disease monitoring ,19,20,23The developed CLIA diagnostic approach detected antibodies in VZV-patient samples with high accuracy/specificity and proportionally to the titer. Interestingly, this approach could detect very low antibody titers and determine positivity in most ELISA-based equivocal results. Moreover, the testing process was simple, fully automated, and thus suitable for high-throughput screening situations. The highly accurate results could be obtained as short as 50 min, with enhanced performance, making this CLIA-based diagnostic a better VZV-diagnostic tool than ELISA. The following The high accuracy demonstrated by the developed diagnostic kit in VZV-specific IgA, IgG, and IgM detection in blood samples was expected. Practically, infection with VZV induces robust antibody response, including IgA, IgG, and IgM antibodies produced mainly against VZV-gE and VZV-gI ,39,40. IThe performance of some current commercially available VZV immunodiagnostic tests, such as VZV TRFIA and VaccZyme™ EIA, has been evaluated and reported . For insThere are a few available diagnostic kits that combine the simultaneous detection of VZV-IgA, IgG, and IgM. Moreover, the diagnostic performance of available IgM ELISA tests is lacking, especially in unvaccinated people ,44,45. OIgM is produced in high titer during the acute phase of primary infection ,40, whilThe use of PCR in VZV diagnosis is preferred and recommended, as it is the most sensitive method to confirm varicella-zoster infection in vesicular lesions or scabs ,24,28. HIn this cohort, analysis of IgA, IgG, and IgM levels showed no significant antibody variation regarding gender (data not shown). However, regarding the age, it came out that while no difference in IgA and IgG level was found, the IgM level showed differences between young adults (below 35) and older. IgM tended to be higher in the youngest than in the eldest, which does not corroborate other studies, in which the antibody titers were proportional with age ,48. A laHowever, although the population size permitted to validate the diagnostic approach, it was not large enough to better evaluate the performance of the test on a representative population scale, precisely to determine the predictive positive and negative values. For the same reason, evaluation of the correlation between each antibody and age range could have been biased as well. Therefore, future investigation with a large and representative population (including vaccinated and non-vaccinated) will better evaluate the performance of this diagnostic approach and study the immune response regarding age range. Moreover, it is suggested to use other samples, including saliva, which is thought to contain higher concentration of antibodies, specifically IgA in VZV infection, for a conclusive added value of IgA detection in the conventional immunodiagnostic kit.In conclusion, detecting VZV-gE-specific IgA, IgG, and IgM using the developed kits based on the CLIA approach provided high sensitivity/accuracy and a rapid practical method for diagnosing VZV in unvaccinated individuals or determining VZV immune status after natural infection. This approach is simple, does not require outstanding trainees, and is suitable in high-throughput diagnosis situations.This study was carried out under the approval (n° 2021-ky269) of the Medical Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (USTC). From June to December 2020, a flow of patients was received in the hospital dermatological department for rashes, pimples, and other skin issues. Based on the presence of typical VZV symptoms , several patients were diagnosed as VZV-infected patients and included after obtaining free consent of participation. Two to three blood samples were collected into EDTA and dry tubes from each enrollee to investigate immunoglobulin (Ig) A, IgG, and IgM in plasma and serum, respectively. ELISA ab108781 (IgA), ab108782 (IgG), and ab108783 (IgM) tests (Abcam) were used as standards to exclude patient samples with negative results for all IgA, IgG, and IgM and retained those with at least one positive/equivocal result for IgA, IgG, or IgM, and thus stratified as true-positive or equivocal group. A total of 29 patients were retained, from which 62 blood samples were obtained for testing the developed CLIA diagnostic approach. Clinical patient data were obtained and listed in Negative control samples were collected to assess the diagnostic accuracy. This cohort contained 453 samples from random healthy consenting people who did not report having suffered from or having been diagnosed positive for VZV infections and did not receive any VZV vaccines. All plasmas and sera were retrieved from EDTA  and dry tubes, respectively, by centrifugation. Retrieved plasmas/sera were treated with 1% TNBP and 1% Triton X-100 to completely denature any potential viruses  and storAs aforementioned, three 96-well plate ELISA tests  were used to detect VZV-specific antibodies (IgA/IgG/IgM) in the enrolled patient samples as a complementary confirmation step. The ELISA tests were performed following the manufacturer’s instructions . Before Patient diagnostic results were determined from OD450 at dilution 1/100, as mentioned in the diagnostic kit leaflet. As a result of considerable background, the OD of the blank (PBS) was deducted from OD450 values of each sample result. Thus, a patient was considered positive when OD450 > 0.2, equivocal when OD450 was between 0.1 and 0.2, and negative when OD450 < 0.1. All patients negative for IgA, IgG, and IgM were systematically excluded from the study, while the retained patients were divided as true-positive or equivocal.To develop the highly-sensitive diagnostic approach detecting VZV-specific IgA, IgG, and IgM in VZV-patient blood samples, the surface antigen glycoprotein E of VZV (VZV-gE) was produced from insect cell cultures using the baculovirus-based vector expression system (BVES) .Spodoptera frugiperda (Sf9) and Trichoplusia ni , were used to produce the baculoviruses carrying the gene of interest and express gE protein, respectively. Sf9 and Hi5 were cultured at 27 °C in SIM-SF and SIM-HF medium , respectively, and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1× penicillin/streptomycin. Sf9 cell lines were maintained in both adherent and suspension cultures, while Hi5 cell lines were only cultured in suspension culture. Adherent cell culture was carried out in 6 cm TC plates, and the suspension cultures were maintained in sterile autoclaved Erlenmeyer flasks in a 110 rpm spin shaker (27 °C). The suspension Hi5 cell culture was diluted to 0.7 to 1 million cells every two based on cell viability and density. The adherents Sf9 cell cultures were detached and diluted every three days based on cell viability and confluence. The cell viability was assessed under a fluorescence microscope using Trypan blue dye (0.4%) and counted using a hemocytometer.Two insect cell lines, including DH10Bac E. coli strain and purified for transfecting Spodoptera frugiperda (Sf9) insect cell lines, which produced recombinant VZV-gE baculoviruses. Expanded recombinant baculovirus stock was used to infect two million Trichoplusia ni  insect cell lines for expressing the recombinant VZV-gE protein. The protein was harvested three days post-infection by high-speed centrifugation and purified from the supernatant.Briefly, the sequence of the mature extracellular region of VZV-gE (GenBank Accession number MH709377.1) was retrieved by PCR from the General Biosystem company’s synthetic construct using the following forward and reverse primers: 5′-ATTTCCAAGGTTCTTCCGTCTTGCGATACGATGATTTTCACATC-3′ and 5′-GACAAGCTTGGTACTTAATATCGTAGAAGTGGTGACGTTCCGGG-3′, respectively. In the meantime, the His-tag-modified transfer vector (pI-SUMO-Star-His) was linearized using primers (forward: 5′CTTCTACGATATTAAGTACCAAGCTTGTCGAGAAGTACTAGAGG3′ and reverse: 5′TATCGCAAGACGGAAGAACCTTGGAAATAAAGATTCTCGCTGCC3′) containing sequences that overlap the VZV-gE 5′ and 3′ end sequences. The linear fragments were ligated following the Gibson Assembly method’s instructions. The successful construct was transposed into bacmid using A couple of purifications steps, including membrane diafiltration (Vivaflow 200), dialysis, ion-nickel column purification, size-exclusion chromatographic purification, and ultra-centrifugation were conducted to purify the protein. The purified protein  was storThe highly purified VZV-gE protein was employed to make the CLIA-based diagnostic kit. The purified VZV-gE protein was first biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin, No-Weigh™ Format kit  following the manufacturer’s instructions. Then, the biotinylated protein was immobilized onto magnetic beads using an Invitrogen Dynabeads™ MyOne™ Streptavidin C1 kit (Thermo Fisher), following the manufacturer’s instructions, and further blocked (or saturated) with 2% of bovine serum albumin (BSA) to avoid non-specific interactions or background. Immobilizing the antigen protein onto a solid phase (here beads) is necessary for immunoblotting IgA, IgG, and IgM antibodies on a solid phase. The detection procedure below was performed with a fully automatic chemical luminescent immune analyzer, Kaeser 1000 . Secondary antibodies anti-human IgA, IgG, or IgM conjugated with acridinium were used to detect the caught VZV-gE specific IgG, IgA, or IgM antibodies, respectively. The detected chemiluminescent signal over the background signal was automatically obtained as relative light units (RLU).These collections, which contain all the described buffers and components for CLIA of VZV gE-specific IgA, IgG, and IgM, are referred to as VZV-gE-IgA, VZV-IgG, and VZV-gE-IgM kits here. As described above, each diagnostic kit was developed independently, with the corresponding secondary antibody conjugated with acridinium.A first test batch of a two-fold serial dilution of the ELISA-based true-positive and healthy plasmas/sera was conducted to assess the reliability of the antibody detection kit regarding sample dilution. A subsequent CLIA test was performed to determine the diagnostic kit performances.Sensitivity (%) = 100 × [True Positive/];Specificity (%) = 100 × [True Negative/];Overall agreement (%) = (True Positive + True Negative)/Total Tests.ELISA tests were triplicate, and the results were transformed, fitted, and presented as mean ± SD. To determine the optimal cut-off values (criteria) and evaluate the diagnostic characteristics of VZV-gE-IgA, IgG, and IgM kits, receiver-operating characteristic (ROC) analyses were performed using MedCalc software. Thus, the specificity and sensitivity of the gE-specific IgA, IgG, and IgM detection kits were determined according to the following formulas:A Mann–Whitney test was used to assess any significant variation of VZV gE-specific IgA, IgG, or IgM level between equivocal and true-positive ELISA-based categories. The same analysis was used to assess any significant correlation of the antibody levels regarding the age ranges. An analysis of variance (ANOVA) test was conducted using the Kruskal–Wallis approach to determine any difference of antibody level between the three independent groups, including positive, equivocal, and negative. A p-value less than 0.05 defined a hypothesis as statistically significant. All the above analyses were integrated into GraphPad Prism5.


To determine whether an increase in cul de sac (CDS) fluid after hysteroscopy is predictive of tubal patency.In a prospective clinical cohort study, 115 subfertile women undergoing laparoscopic and hysteroscopic surgery at the Medical University of Vienna were invited to participate. The primary outcome was determining whether an increase in fluid in the pouch of Douglas was reflective of unilateral or bilateral tubal patency. Vaginal sonography before and after hysteroscopy was performed to detect fluid in the pouch of Douglas, directly followed by laparoscopy with chromopertubation.p < 0.001). One/75 patients (1.3%) showing a fluid shift had bilateral occlusion . Intracavitary abnormalities  and adhesions covering one or both tubes  increased the risk for a false abnormal result, i.e., uni- or bilateral tubal patency despite the lack of a fluid shift.Laparoscopic chromopertubation revealed bilateral Fallopian tube occlusion in 28 women (24.3%). Twenty-seven/40 patients (67.5%) with no fluid shift had bilateral occlusion during the consecutive laparoscopy (When CDS fluid does not change after hysteroscopy, this is a sensitive test for tubal occlusion and further testing may be warranted. However, if there is an increase in CDS fluid after hysteroscopy, particularly for a patient without fluid present prior, this is both sensitive and specific for unilateral or bilateral tubal patency. Hysteroscopy is known as the gold standard for intrauterine evaluation , 2 and cNeither of these studies, in describing finding studies, took all patients to the operating room. For the Peregrin-Alvarez study, no patients went to the operating room, and for the Parry, et al., study, though an association was described between occlusion at surgery and a lack of increase in CDS fluid after hysteroscopy, the associated sensitivity and specificity was not published. Since there are few publications on this issue, our research goal was to better understand how tubal patency affects CDS fluid shifts through a prospective cohort study.https://data.mendeley.com/datasets/zf3hp5myyg/1).In a prospective, monocentral, clinical cohort study, 115 women ages 18–44 were enrolled between March 2018 and November 2019. Participants were undergoing a fertility evaluation at the Clinical Division of Gynecologic Endocrinology and Reproductive Medicine of the Medical University of Vienna, Austria. All participants signed written affirmation of informed consent prior to a combination of hysteroscopy and laparoscopy with chromopertubation. Vaginal sonography was performed before and after the hysteroscopy. Patients who had already removed one or both of their Fallopian tubes were excluded from participation in the study. The study was approved by the Institutional Review Board of the Medical University of Vienna (IRB number 1096/2018). The dataset is available online  was placed through the cervix before laparoscopy with chromopertubation.A “Spackmann” uterine manipulator with clamp fixation and a rubber cone with a diameter of 18 mm  was performed in all patients pre-hysteroscopy and post-hysteroscopy to assess shifts in fluid volume for the pouch of Douglas. The fluid pockets located in the vaginal rear fornix were measured, as previously characterized . SpecifiThe parameters assessed were documented by the senior surgeon using a prospective case report form. We considered the presence of a sonographic fluid shift in Douglas pouch before and after the hysteroscopy to be the main outcome parameter. These findings were validated by laparoscopic chromopertubation. Furthermore, hysteroscopically diagnosed abnormalities in the uterine cavity, tubal patency in chromopertubation (for each side) and information about further laparoscopic surgical interventions were considered additional outcome parameters. Outcomes were always supervised by a study member who was not part of the surgical team for each respective operation. In addition, tracked parameters include full uterine length, patient’s age and body mass index (BMI), the type of infertility (primary versus secondary), the surgical indication, and the menstrual cycle day at surgical date.A similar study about transvaginal sonography for the detection of hysteroscopic fluid in Douglas pouch assessed a group with lower risk for surgical pathology  relative®-table. Data analyses were conducted using SPSS software 24.0 . Numerical data with a normal distribution were reported as mean and standard deviations, numerical data without a normal distribution as median and interquartile range (IQR), while categorical data  were reported as number and percentage.The AKIM software  was used for data acquisition, after data were collected and documented via prospective case report form and an Excelp < 0.05.Differences between groups in categorical parameters were tested using the Chi square test or the Fisher’s exact test. Overall accuracy, sensitivity, specificity, positive and negative predictive values including the 95% confidence intervals (95% CI) were calculated for presence of a sonographic cul de sac fluid shift for the prediction of bilateral tubal occlusion. Risk factors for false-normal results were evaluated. For this, a multivariate binary regression model was used. This analysis used odds ratios with 95% confidence intervals (95% CI), the Wald-test, and the likelihood ratio (LR) test. Differences were considered significant if Patient characteristics are provided in Table In subsequent laparoscopic chromopertubation, there were 152 (66.1%) patent Fallopian tubes  and 78 (33.9%) occluded Fallopian tubes . Chromopertubation revealed a unilateral tubal occlusion in 22 (19.1%) and bilateral tubal occlusion in 28 (24.3%) women. Notably, in 11/22 (50.0%) patients with unilateral occlusion, high chromopertubation pressure was necessary to achieve patency. For the 50 women with unilateral or bilateral Fallopian tube occlusion, 26 (52%) related to endometriosis and/or hydrosalpinges, while neither of these were identified in 24 (48%).p = 0.002). After hysteroscopy, cul de sac fluid was found in 80/115 patients (69.6%), with a median volume of 15.5 mL (IQR 5.3–30.0 mL). However, a clearly visible fluid shift was identified for 75 women (65.2%). Women with bilateral tubal patency revealed a significantly higher increase in cul de sac fluid than women with unilateral tubal occlusion and women with bilateral tubal occlusion . In these women, a median amount of 3.4 mL IQR 2.5–6.0 mL) was found. This was more often the case in the luteal phase  compared to the follicular phase . Twenty-seven of 40 patients (67.5%) for whom no fluid shift was identified had bilateral occlusion during the subsequent laparoscopy, whereas this was the case for only 1/75 patients (1.3%) showing a visible fluid shift, which results in a NPV of 67.5% (95% CI: 50.9–81.4) and a PPV of 98.7% (95% CI: 92.8–100.0%). The one patient with a sonographic fluid shift despite bilateral tubal occlusion at laparoscopy may have been a false positive with chromopertubation. She was a 34 years old woman who underwent the operation for primary infertility, suspicion of an endometrial polyp and suspicion of endometriosis (both of which were confirmed). During laparoscopy, a bilateral proximal tubal occlusion was diagnosed as well as endometriosis of rASRM stage 3 (without endometriomas). The sonographically measured volume of fluid in the pouch of Douglas had increased from 2.4 mL pre-hysteroscopy to 15.0 mL post-hysteroscopy. About 2 months after the operation, a hysterosalpingo-contrast-sonography was performed in an outpatient setting and revealed bilateral tubal patency, suggesting that chromopertubation had improperly identified bilateral occlusion.As demonstrated in Table p < 0.001). In other words, of 40 women without a fluid shift, only 3 (7.5%) had bilateral tubal patency. The associated sensitivity was 74% (95% CI: 59.7–85.4%) and specificity 95.4% (95% CI: 87.1–99.0%). Of the ten women with unilateral occlusion and without a fluid shift, at laparoscopy, seven (70.0%) revealed peritubal adhesions on the patent side.We also tested whether the presence of a fluid shift would bilateral tubal patency Table B. Of then = 27, 67.5%) to those without  bilateral Fallopian tube occlusion (Table p = 0.030) or adhesions covering one or both tubes . When chromopertubation was performed demonstrating patency, it was after correction of intrauterine pathology in 7/13 women (53.8%), whereas in 6/13 women (46.2%) with neither a fluid shift nor bilateral Fallopian tubal occlusion, neither intracavitary abnormalities nor adhesions covering one or both tubes were found.In addition, we did subset analysis in the 40 patients for whom no fluid shift had been seen and compared those with  in our patient population. Thus, for the first time, tubal spasm during laparoscopic chromopertubation can be defined and diagnosed, at least those cases that occur after hysteroscopy. Accordingly, we believe that performing a vaginal ultrasound directly before and after hysteroscopy might also be a valuable tool in infertile women who undergo hysteroscopy and subsequent laparoscopy with chromopertubation. It has to be noted that this additional procedure can be performed easily and is time efficient.Given reasonable sensitivity, specificity, positive, and negative predictive values, the presence and absence of fluid shifts in the pouch of Douglas can provide valuable insight for tubal patency in patients undergoing hysteroscopy during a procreative workup. Moreover, cul de sac fluid shifts can be combined with observational (such as the flow technique ) and intThe strength of this study is that all included women underwent laparoscopic chromopertubation which allowed comparison of the sonographic results to the gold standard. Moreover, additional data about endometriosis, adhesions and other intraabdominal abnormalities could be evaluated. However, a few study limitations have to be mentioned. One might argue that the rate of women with a cloudy view in hysteroscopy was high (23.5%). However, a previous study has demonstrated that this finding was associated with bilateral tubal occlusion  which waSonographically observing pre- to post-hysteroscopic fluid shifts in the pouch of Douglas offers sensitive and specific information about tubal patency and occlusion, which can be useful in counseling patients with subfertility. The absence of a fluid shift can be suggestive of both unilateral and bilateral tubal occlusion, where women with these findings likely should undergo additional tubal patency assessment. In addition, women with increases in cul de sac fluid volume, particularly if they intend to try to conceive naturally, can be reassured that they likely have unilateral or bilateral patency. Moreover, when patients have indeterminate results through direct hysteroscopic assessment, such as the flow and Parryscope techniques, concurrent ultrasound can often still allow for enough guidance as to make decisions on next steps, particularly when accounting for known risk factors. Particularly when combining hysteroscopy and sonography in the office, this minimally invasive approach to the fertility evaluation may offer meaningful information for patients.