A novel homozygous LRRC6 mutation causes male infertility with asthenozoospermia and primary ciliary dyskinesia in humans.

BACKGROUND: Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes. OBJECTIVES: To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved. MATERIALS AND METHODS: The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations. RESULTS: In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer dynein arms and inner dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband. DISCUSSION AND CONCLUSION: This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.

Novel variants in TUBB8 gene cause multiple phenotypic abnormalities in human oocytes and early embryos.

BACKGROUND: The genotype-phenotype relationships between TUBB8 variants and female infertility are difficult to clearly define due to the complex inheritance patterns and the highly heterogeneous phenotypes. This study aims to identify novel TUBB8 variants and relevant phenotypes in more infertile females. METHODS: A total of 35 females with primary infertility were recruited from two reproductive centers and investigated for identifying variants in TUBB8. Pedigree analysis, in-silico analysis and molecular remodeling were performed to assess their clinical significance. The effects of the variants on human oocytes and embryos as well as HeLa cells were analyzed by morphological observations, immunostaining and Western blot. RESULTS: We totally identified five novel variants (p.G13R, p.Y50C, p.T136I, p.F265V and p.T366A) and five previously reported variants (p.I4L, p.L42V, p.Q134*, p.V255M and p.V349I) in TUBB8 from 9 unrelated females with primary infertility. These variants were rare and highly conserved among different species, and were inherited in autosomal dominant/recessive patterns, or occurred de novo. In vitro functional assays in HeLa cells revealed that exogenous expression of mutant TUBB8 proteins caused different degrees of microtubule structural disruption. The existence of these pathogenic TUBB8 variants finally induced oocyte maturation arrest or morphological abnormalities, fertilization failure, cleavage failure, embryonic development defects and implantation failure in the affected females. CONCLUSION: These findings enriched the variant spectrum of TUBB8 gene and could contribute to optimize genetic counselling and clinical management of females with primary infertility.

Molecular Classification in Patients With Endometrial Cancer After Fertility-Preserving Treatment: Application of ProMisE Classifier and Combination of Prognostic Evidence.

The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) is a molecular classification system that identifies endometrial cancer (EC) into four prognostically distinct subtypes: POLE-mutated, mismatch repair deficiency (MMR-D), p53 wild-type (p53wt), and p53 abnormal (p53abn). However, few reports have applied the ProMisE classifier to EC patients who underwent fertility-preserving treatment (FPT) so far. This study evaluated whether the ProMisE classifier predicted in early-stage EC patients after FPT. We first summarized the three reported outcomes of ProMisE applied to EC patients who received FPT. The hormone-treated patients with EC from 2010 to 2020 in our facility were then analyzed. By sequential immunohistochemistry and Sanger sequencing of POLE according to the ProMisE system, formalin-fixed paraffin-embedded blocks of patients before treatment were collected and classified into POLE-mutated, MMR-D, p53wt, and p53abn subtypes. The primary outcome was a complete response rate after FPT. Thirteen patients were enrolled from our facility, with 3 (3/13) MMR-D, 0 (0/13) POLE, 8 (8/13) p53wt, 1 (1/13) p53abn, and 1 (1/13) failed with DNA amplification. Six (6/8) patients with p53wt, 2 (2/3) patients with MMR-D, and 1 (1/1) patient with p53abn achieved a complete response in 6 months after treatment. The results of our study and the reported outcomes were finally combined. A total of 106 patients who underwent FPT were included. Of these, 23 (21.7%) were classified as MMR-D, 3 (2.8%) as POLE-mutated, 3 (2.8%) as p53abn, and 77 (72.6%) as p53wt. There was no significant difference in the complete response rate (P = 0.152) and recurrence rate (P = 0.174) between MMR-D and p53wt subtypes after FPT. Based on current data, we observed no prognostic significance of the ProMisE classifier in EC patients who underwent FPT. Larger prospective studies are needed to elucidate the precise prognostic meaning of this molecular classifier in these cases.

REBACIN® is an optional intervention for persistent high-risk human papillomavirus infection: A retrospective analysis of 364 patients.

OBJECTIVE: To evaluate the therapeutic efficacy of REBACIN® in patients with persistent high-risk HPV (hrHPV) infection. Persistent hrHPV infection is a crucial cause of cervical cancer, for which optimal pharmacological intervention remains unavailable. METHODS: A retrospective analysis and a meta-analysis were carried out. The retrospective analysis included 364 patients who were persistently infected with HPV for at least 12 months, between September 2015 and February 2019, and only received the REBACIN® intervention. HPV DNA typing, HC2 hrHPV DNA, and ThinPrep cytologic tests were performed before and after the REBACIN® intervention, to evaluate the therapeutic efficacy. The meta-analysis included trials evaluating the therapeutic efficacy of interferons. RESULTS: After a follow-up period of 3-6 months, the overall rate of efficacy of REBACIN® was 74.73% (272/364), which was higher than that of interferon (61.50%). The efficacy of REBACIN® was correlated with HPV type (odds ratio [OR] 0.549, 95% confidence interval [CI] 0.367-0.822, P=0.004) and pretreatment cytology (OR 0.358, 95% CI 0.173-0.739, P=0.005). CONCLUSION: REBACIN® is potently efficacious at clearing persistent hrHPV infection; hence, it can serve as an optional intervention for persistent hrHPV infection.

Role of epithelial-mesenchymal transition regulated by twist basic helix-loop-helix transcription factor 2 (Twist2) in embryo implantation in mice.

In a previous study we found the expression of epithelial-mesenchymal transition (EMT) biomarkers, including E-cadherin and N-cadherin, was significantly altered in uterine endometrium during embryo implantation via regulation by microRNA (miRNA)-429 and protocadherin-8 (Pcdh8). As a natural continuation of the previous study, the aim of the present study was to explore the role of EMT during embryo implantation and the potential activity of twist basic helix-loop-helix transcription factor 2 (Twist2) in regulating embryo implantation. A pregnancy model was established by naturally mating adult female ICR mice with fertile males. A pseudopregnancy model was established by mating fertile female ICR mice with vasectomised males. An invitro model of embryo implantation was established by the coculture of Ishikawa and JAR spheroids. Endometrial tissue during the peri-implantation period was collected, as were Ishikawa cells, JAR cells and cocultured cells. The expression of EMT markers (E-cadherin, N-cadherin, vimentin and cytokeratin) and Twist2 was detected invivo and invitro using the western blot analysis during embryo implantation. The expression of N-cadherin and vimentin (mesenchymal markers) was upregulated in the invitro implantation model, with downregulation of E-cadherin and cytokeratin (epithelial markers) expression. The expression of N-cadherin, vimentin and Twist2 increased significantly at the implantation sites at the time of implantation (Day 5), whereas the expression of E-cadherin and cytokeratin decreased. Location of Twist2 during embryo implantation was detected by immunohistochemistry (IHC), which revealed that it was extensively expressed in endometrial glandular epithelium and luminal epithelium at implantation sites on Day 5. The effect of the expression of Twist2 on embryo implantation was evaluated by suppressing Twist2 using Twist2-short interference (si) RNA in invivo and invitro models. The numbers of implanted embryos and the implantation rate were compared invivo and invitro. Western blot analysis showed that suppression of Twist2 led to upregulation of E-cadherin and cytokeratin, accompanied by downregulation of N-cadherin and vimentin (P<0.05). The number of implanted embryos after Twist2-siRNA interference was lower than in normal pregnancy (mean (±s.d.) 2.4±0.5 vs 6.8±1.3 respectively; P<0.05). These findings suggest the involvement of EMT in embryo implantation. The suppression of Twist2 could suppress embryo implantation by regulating EMT.