Severe asthenozoospermia is a common cause of male infertility. Recent studies have revealed that
SPEF2
mutations lead to multiple morphological abnormalities of the sperm flagella (MMAF) without ...primary ciliary dyskinesia (PCD) symptoms in males, but PCD phenotype was also found in one female individual. Therefore, whether there is a phenotypic continuum ranging from infertile patients with PCD to MMAF patients with no or low noise PCD manifestations remains elusive. Here, we performed whole-exome sequencing in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. We identified four novel biallelic mutations in
SPEF2
(8.9%, 4/45) in six affected individuals (12.8%, 6/47), while no deleterious biallelic variants in
SPEF2
were detected in 637 controls, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia, and 223 fertile controls. Notably, all six patients exhibited PCD-like symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis. Ultrastructural analysis revealed normal 9 + 2 axonemes of respiratory cilia but consistently abnormal 9 + 0 axoneme or disordered accessory structures of sperm flagella, indicating different roles of SPEF2 in sperm flagella and respiratory cilia. Subsequently, a
Spef2
knockout mouse model was used to validate the PCD-like phenotype and male infertility, where the subfertility of female
Spef2
−/−
mice was found unexpectedly. Overall, our data bridge the link between MMAF and PCD based on the association of
SPEF2
mutations with both infertility and PCD in males and provide basis for further exploring the molecular mechanism of SPEF2 during spermiogenesis and ciliogenesis.
Comorbid familial nonobstructive azoospermia (NOA) and congenital cataract (CC) have not been reported previously, and no single human gene has been associated with both diseases in humans. Our ...purpose was to uncover novel human mutations and genes causing familial NOA and CC.
We performed whole-exome sequencing for two brothers with both NOA and CC from a consanguineous family. Mutation screening of TDRD7 was performed in another similar consanguineous family and 176 patients with azoospermia or CC alone and 520 healthy controls. Histological analysis was performed for the biopsied testicle sample in one patient, and knockout mice were constructed to verify the phenotype of the mutation in TDRD7.
Two novel loss-of-function mutations (c.324_325insA (T110Nfs*30) and c.688_689insA (p.Y230X), respectively) of TDRD7 were found in the affected patients from the two unrelated consanguineous families. Histological analysis demonstrated a lack of mature sperm in the male patient's seminiferous tubules. The mutations were not detected in patients with CC or NOA alone. Mice with Tdrd7 gene disrupted at a similar position precisely replicated the human syndrome.
We identified TDRD7 causing CC as a new pathogenic gene for male azoospermia in human, with an autosomal recessive mode of inheritance.
Disturbances in maternal lipid metabolism may increase the risk of developing pregnancy complications and adverse perinatal outcomes. However, there is no consensus as to what constitutes normal ...serum lipid ranges during pregnancy. Our study was aimed to establish trimester-specific serum lipid reference intervals (RIs) and investigate the associations between maternal dyslipidaemia and adverse outcomes in a population-based study.
The first- and third-trimester lipid profiles were derived from 16,489 singlet pregnant women for regular antenatal check-ups between 2017 and 2019. The serum samples were assayed for total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) in the institutional clinical laboratory. The trimester-specific lipid RIs were estimated with both of the direct observational and the indirect Hoffmann methods. The associations between maternal lipid profiling and pregnancy complications and perinatal outcomes were assessed statistically.
Serum levels of TC, TG, LDL-C and HDL-C were all increased significantly in the third trimester of pregnancy. There was no significant difference between the observed RIs established with healthy pregnant women and the calculated RIs derived from the Hoffmann method. A trend towards increased risks of gestational complications and adverse perinatal outcomes was observed in the subjects with elevated levels of TC, TG, and LDL-C or decreased level of HDL-C.
In pregnancy, increased serum levels of TC, TG and LDL-C, and a decreased level of HDL-C posed higher risks of developing pregnancy complications and adverse perinatal outcomes.
Key messages
It is necessary to establish trimester-specific reference intervals for serum lipids including TC, TG, LDL-C and HDL-C that were found significantly increased as the gestational age went up. More importantly, around the upper reference limits of TC, TG and LDL-C (or the lower reference limit of HDL-C), the higher the serum lipid levels were (or the lower the HDL-C level was), the higher risks of developing pregnancy complications and adverse perinatal outcomes were observed.
BACKGROUNDAs folates are essential for embryonic development and growth, it is necessary to accurately determine the levels of folates in plasma and red blood cells (RBCs) for clinical intervention. ...The aims of this study were to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of folates in plasma and RBCs and to examine the association between plasma and RBC folate concentrations and gestational diabetes mellitus (GDM), gestational hypertension (GH) and preeclampsia (PE).METHODSWith the in-house developed LC-MS/MS, a retrospective cross-sectional study was conducted. The healthy pregnant women of first- (n = 147), second- (n = 84) and third-trimester (n = 141) or the women diagnosed with GDM (n = 84), GH (n = 58) or PE (n = 23), that were aged between 22 and 46 years old and registered at our institute, were subjected for measurement of folic acid (FA) and 5-methyltetrahydrofolate (5-MTHF), followed by appropriate statistical association analysis.RESULTSThe assay for simultaneous quantitation of FA and 5-MTHF in plasma and RBCs was linear, stable, with imprecision less than 15% and recoveries within ±10%. The lower limits of quantification for FA and 5-MTHF measurement in whole blood were 0.57 and 1.09 nmol/L, and in plasma were 0.5 and 1 nmol/L, respectively. In the association analysis, the patients with lower RBC folate level (<906 nmol/L) presented higher risks of PE development (OR 4.861 95% CI 1.411-16.505) by logistic regression and restricted cubic spline (RCS) regression in a nonlinear fashion. In addition, higher level of plasma folates in pregnancy was significantly associated with GH risk but may be protective for the development of GDM.CONCLUSIONSThe in-house developed LC-MS/MS method for folates and metabolites in plasma or RBC showed satisfactory analytical performance for clinical application. Further, the levels of folates and metabolites were diversely associated with GDM, GH and PE development.
Prediction of delivery is important for assessing due dates, providing adequate prenatal care, and suggesting appropriate interventions in preterm and post-term pregnancies. Recent metabolomic ...findings suggested that the temporal abundance information of metabolome can be used to predict delivery timing with high accuracy in a cohort of healthy women. However, a targeted and quantitative assay is required to further validate the clinical performance and utility of this group of metabolomic candidates in delivery prediction with a larger and independent cohort.
LC-MS/MS quantitative assays were applied to determine the plasma concentrations of four steroid metabolites, including oestriol-16-glucuronide (E3-16-Gluc), 17-alpha-hydroxyprogesterone (17-OHP), tetrahydrodeoxycorticosterone (THDOC), and androstane-3,17-diol (A-3,17-Diol) in asymptomatic women of singleton pregnancies (≥30
th
gestational weeks). Subsequent statistical analysis was conducted to assess the performance of the above candidates in delivery prediction.
Using LC-MS/MS, four steroids were separated and quantified in 5.5 min. The coefficients of variation (CVs) of the four analytes at the lower limit of quantification ranged from 7.9% to 14.6%, with the R
2
values greater than 0.990 in the calibration curves. Of the 585 recruited pregnant women who ended up with spontaneous delivery, 17.1% and 82.9% of the subjects delivered within and after 7 days since plasma collection, respectively. In the receiver operator curve analysis, the gestational age-adjusted area under the curve of the combined measurements of E3-16-Gluc and 17-OHP was 0.69 (95% CI: 0.60-0.76), with the sensitivity of 87.0% (95% CI: 78.8%-92.9%) and specificity of 60.2% (95% CI: 55.7%-64.6%). Moreover, the positive and the negative predictive values were 28.3%-34.0% and 93.1%-97.4% respectively for this combined panel.
We performed analytical and clinical validation of a quantitation LC-MS/MS panel for the four steroids in the plasma of pregnant women. The steroid metabolites panel of E3-16-Gluc and 17-OHP was potentially useful for predicting delivery within one week in asymptomatic women of singleton pregnancies.
Key messages
A quantitative LC-MS/MS assay for determining the plasma levels of 17-OHP, THDOC, A-3,17-Diol and E3-16-Gluc was developed and validated, in order to evaluate their predictive performance in asymptomatic delivery of singleton pregnancy. The levels of E3-16-Gluc and 17-OHP were found to be significantly elevated at the time of sampling in women that delivered within one week and their combinational testing may be potentially useful in delivery prediction.
Purpose
To identify the genetic cause of patients with primary ciliary dyskinesia (PCD) and male infertility from two unrelated Han Chinese families.
Methods
We conducted whole-exome sequencing of ...three individuals with PCD and male infertility from two unrelated Chinese families, and performed a targeted look-up for
DNAAF6
variants in our previously reported cohort of 442 individuals (219 with isolated oligoasthenospermia and 223 fertile controls). Ultrastructural and immunostaining analyses of patients’ spermatozoa were performed. The pathogenicity of the variants was validated using patient’s spermatozoa and HEK293T cells. Intracytoplasmic sperm injection (ICSI) treatment was conducted in two patients.
Results
We identified one novel hemizygous frameshift variant (NM_173494, c.319_329del: p.R107fs) of
DNAAF6
gene (previously named
PIH1D3
) in family 1 and one novel hemizygous missense variant (c.290G>T: p.G97V) in family 2. No hemizygous deleterious variants in
DNAAF6
were detected in the control cohort of 442 individuals. Ultrastructural and immunostaining analyses of patients’ spermatozoa showed the absence of outer and inner dynein arms in sperm flagella. Both variants were proven to lead to DNAAF6 protein degradation in HEK293T cells. Both patients carrying
DNAAF6
variants underwent one ICSI cycle and delivered one healthy child each.
Conclusion
We identified novel
DNAAF6
variants causing male infertility and PCD in Han Chinese patients. This finding extended the spectrum of variants in
DNAAF6
and revealed new light on the impact of
DNAAF6
variants in sperm flagella.
Background:
Infertility is a global health concern.
MEIOB
has been found to be associated with premature ovarian insufficiency (POI) and non-obstructive azoospermia (NOA), but its variants have not ...been reported in Chinese patients. The aim of this study was to identify the genetic aetiology of POI or NOA in three Han Chinese families.
Methods:
Whole-exome sequencing (WES) was used to identify candidate pathogenic variants in three consanguineous Chinese infertile families with POI or NOA. Sanger sequencing was performed to validate these variants in the proband of family I and her affected family members.
In vitro
functional analyses were performed to confirm the effects of these variants.
Results:
Two novel homozygous frameshift variants (c.258_259del and c.1072_1073del) and one novel homozygous nonsense variant (c.814C > T) in the
MEIOB
gene were identified in three consanguineous Han Chinese families.
In vitro
functional analyses revealed that these variants produced truncated proteins and affected their function.
Conclusion:
We identified three novel
MEIOB
loss-of-function variants in local Chinese patients for the first time and confirmed their pathogenicity using
in vitro
functional analyses. These results extend the mutation spectrum of the
MEIOB
gene and have important significance for genetic counselling in these families.
Inner dynein arm (IDA), composed of a series of protein complex, is necessary to cilia and flagella bend formation and beating. Previous studies indicated that defects of IDA protein complex result ...in multiple morphological abnormalities of the sperm flagellum (MMAF) and male infertility. However, the genetic causes and molecular mechanisms in the IDAs need further exploration. Here we identified two loss-of-function variants of WDR63 in both MMAF and non-obstructive azoospermia (NOA) affected cohorts. WDR63 encodes an IDA-associated protein that is dominantly expressed in testis. We next generated Wdr63-knockout (Wdr63-KO) mice through the CRISPR-Cas9 technology. Remarkably, Wdr63-KO induced decreased sperm number, abnormal flagellar morphology and male infertility. In addition, transmission electron microscopy assay showed severely disorganized "9 + 2" axoneme and absent inner dynein arms in the spermatozoa from Wdr63-KO male mice. Mechanistically, we found that WDR63 interacted with WDR78 mainly via WD40-repeat domain and is necessary for IDA assembly. Furthermore, WDR63-associated male infertility in human and mice could be overcome by intracytoplasmic sperm injection (ICSI) treatment. In conclusion, the present study demonstrates that bi-allelic variants of WDR63 cause male infertility via abnormal inner dynein arms assembly and flagella formation and can be used as a genetic diagnostic indicator for infertility males.
A ZnCl2‐promoted one‐pot synthesis of multisubstituted thiazolo4,5‐bpyridines and thieno2,3‐b:4,5‐b′dipyridines from mercaptonitrile salts, α‐bromo ketones and ketones has been developed. This ...approach involves a tandem SN2 alkylation/Thorpe–Ziegler cyclization/Friedländer reaction, which allows the creation of two hetero rings and four new bonds in a single operation. The reaction proceeds smoothly under solvent‐free conditions to afford fused pyridines in moderate to good yields.
A facile ZnCl2‐promoted one‐pot three‐component synthesis of thiazolo4,5‐bpyridines and thieno2,3‐b:4,5‐b′dipyridines is described. This approach involves a tandem SN2/Thorpe–Ziegler cyclization/Friedländer reaction to give fused pyridines in moderate to good yields.
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