Two missense STK11 gene variations impaired LKB1/adenosine monophosphate-activated protein kinase signaling in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11 (STK11/LKB1) gene mutations. Preimplantation genetic testing can protect a patient's offspring from mutated genes; however, some variations in this gene have been interpreted as variants of uncertain significance (VUS), which complicate reproductive decision-making in genetic counseling. AIM: To identify the pathogenicity of two missense variants and provide clinical guidance. METHODS: Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya. Software was employed to predict the protein structure, conservation, and pathogenicity of the two missense variation sites in patients with PJS. Additionally, plasmids were constructed and transfected into HeLa cells to observe cell growth. The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry. Statistical analysis was performed using one-way analysis of variance. P < 0.05 was considered statistically significant. RESULTS: We identified two missense STK11 gene VUS [c.889A>G (p.Arg297Gly) and c.733C>T (p.Leu245Phe)] in 9 unrelated PJS families who were seeking reproductive assistance. The two missense VUS were located in the catalytic domain of serine/threonine kinase, which is a key structure of the liver kinase B1 (LKB1) protein. In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells. In addition, the two missense STK11 variants promoted the proliferation of HeLa cells. Subsequent immunohistochemical analysis showed that phosphorylated-AMPK (Thr172) expression was significantly lower in gastric, colonic, and uterine polyps from PJS patients with missense variations than in non-PJS patients. Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene, causing it to lose its function of regulating downstream phosphorylated-AMPK (Thr172), which may lead to the development of PJS. The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance. CONCLUSION: These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients. These findings not only offer insights for clinical decision-making, but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.

A hemizygous loss-of-function variant in BCORL1 is associated with male infertility and oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.

A case report of a normal fertile woman with 46,XX/46,XY somatic chimerism reveals a critical role for germ cells in sex determination.

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction.

Bi-allelic variants in DNAH3 cause male infertility with asthenoteratozoospermia in humans and mice.

STUDY QUESTION: Are there other pathogenic genes for asthenoteratozoospermia (AT)? SUMMARY ANSWER: DNAH3 is a novel candidate gene for AT in humans and mice. WHAT IS KNOWN ALREADY: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. STUDY DESIGN SIZE DURATION: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. MAIN RESULTS AND THE ROLE OF CHANCE: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.

Mosaic variegated aneuploidy syndrome with tetraploid, and predisposition to male infertility triggered by mutant CEP192.

In this study, we report on mosaic variegated aneuploidy (MVA) syndrome with tetraploidy and predisposition to infertility in a family. Sequencing analysis identified that the CEP192 biallelic variants (c.1912C>T, p.His638Tyr and c.5750A>G, p.Asn1917Ser) segregated with microcephaly, short stature, limb-extremity dysplasia, and reduced testicular size, while CEP192 monoallelic variants segregated with infertility and/or reduced testicular size in the family. In 1,264 unrelated patients, variant screening for CEP192 identified a same variant (c.5750A>G, p.Asn1917Ser) and other variants significantly associated with infertility. Two lines of Cep192 mice model that are equivalent to human variants were generated. Embryos with Cep192 biallelic variants arrested at E7 because of cell apoptosis mediated by MVA/tetraploidy cell acumination. Mice with heterozygous variants replicated the predisposition to male infertility. Mouse primary embryonic fibroblasts with Cep192 biallelic variants cultured in vitro showed abnormal morphology, mitotic arresting, and disruption of spindle formation. In patient epithelial cells with biallelic variants cultured in vitro, the number of cells arrested during the prophase increased because of the failure of spindle formation. Accordingly, we present mutant CEP192, which is a link for the MVA syndrome with tetraploidy and the predisposition to male infertility.

Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure.

ABSTRACT: Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca2+ oscillations and the subsequent block to polyspermy following gamete fusion. Mutations in PLCZ1, the gene encoding PLCζ, cause male infertility and intracytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate (FR). However, in conventional in vitro fertilization (cIVF), whether and how sperm PLCζ affects fertilization remain unclear. Herein, we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei (PN) formation. Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca2+ oscillations. ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Furthermore, we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between February 2019 and January 2022. We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR. The optimal cutoff value below which males were likely to experience low FR (total FR ≤30%) after cIVF was 56.7% for the proportion of spermatozoa expressing PLCζ. Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the wider male population and not only men with ICSI failure.

Immune profiling and RNA-seq uncover the cause of partial unexplained recurrent implantation failure.

BACKGROUND: Detailed knowledge of the changes in endometrial immune cells during the window of implantation in unexplained recurrent implantation failure (RIF) patients, the functions performed by immune cells, and the interactions between them is largely lacking. This study aimed to classify RIF patients and explore the mechanism through endometrial immune profiling and RNA-seq analysis. METHODS: This study enrolled a total of 172 patients, comprising 144 women with unexplained RIF and 28 fertile women. Endometrial samples were collected using endometrial scratching at the mid-luteal phase before in vitro fertilization treatment or pregnancy. Transcriptome sequencing and immunohistochemical staining of endometrial immune cells including natural killer (NK) cells, macrophages, T cells, and B cells were performed. MAIN OUTCOME MEASURE(S): Comparison of the percentage of endometrial immune cells and the RNA-seq information between RIF patients and fertile control patients. RESULT(S): The proportions of uterine CD56+ uNK cells, CD57+ NKT cells, CD68+ macrophages, and CD19+ B cells were significantly elevated in RIF patients. In addition, the number of positive CD68 glandular lumens was significantly higher in RIF patients than in the fertile group. In addition, based on this result, we classified RIF patients into three categories. CONCLUSION(S): Hyperactivation of endometrial immune cells may be associated with reduced endometrial tolerance and recurrent implantation failure, affecting pregnancy outcomes in RIF patients.

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

STUDY QUESTION: What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? SUMMARY ANSWER: Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. WHAT IS KNOWN ALREADY: The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. STUDY DESIGN, SIZE, DURATION: Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. MAIN RESULTS AND THE ROLE OF CHANCE: Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. LIMITATIONS, REASONS FOR CAUTION: More cases are needed to demonstrate the relation between IQCN variants and phenotypes. WIDER IMPLICATIONS OF THE FINDINGS: Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER: N/A.

Histological endometrial dating: a reliable tool for personalized frozen-thawed embryo transfer in patients with repeated implantation failure in natural cycles.

OBJECTIVE: To evaluate the clinical availability and stability of histological endometrial dating as a tool for personalized frozen-thawed embryo transfer (pFET) in patients with repeated implantation failure (RIF) in natural cycles. METHODS: A total of 1245 RIF patients were recruited to the present study. All of the patients received an endometrial dating evaluation on day 7 post-ovulation (PO + 7) to guide their first pFET. The second and third pFETs were executed according to histological examination (again employing biopsy) or by reference to previous results. Subsequent pregnancy outcomes for all of the cycles were ultimately tracked. RESULTS: The out-of-phase rate for RIF patients was 32.4% (404/1245) and the expected dating rate (the probability of the expected endometrial dating aligning with repeat biopsy) for endometrial dating reevaluation was as high as 94.3% (50/53). The clinical pregnancy rates of first, second, and third pFETs were 65.3%, 50.0%, and 44.4%, respectively; and the cumulative clinical pregnancy rate attained 74.9% after three transfers. Endometrial dating reevaluations met expectations with more than a 2-year duration in three cases and elicited favorable clinical outcomes. CONCLUSION: We validated the relatively high stability of the histological endometrial dating platform-including the out-of-phase rate and the expected dating rate of reevaluation in patients with RIF-by expanding the sample size. The pFET, based on histological endometrial dating, was of acceptable clinical value and was worthy of promotion in patients with unexplained RIF.

A DMD case caused by X chromosome rearrangement.

Duchenne/Becker muscular dystrophy (DMD/BMD) is one of the most common progressive muscular dystrophy diseases with X-linked recessive inheritance. It is mainly caused by the deletion, duplication and point mutation of DMD gene. In rare cases, it is also caused by the destruction of DMD gene by chromosomal structural rearrangement. Here, we report a case of Duchenne/Becker Muscular dystrophy (DMD/BMD) with typical symptoms but unknown genetic defects after MLPA and next generation sequencing tests in other hospitals. Interestingly, we find a pericentric inversion of X chromosome (Chr.X: g. [31939463-31939465del; 31939466-131765063 inv; 131765064-131765067del]) in this patient. We then use the karyotyping, FISH, long-read sequencing and Sanger sequencing technologies to characterize the chromosome rearrangement. We find that this chromosomal aberration disrupt both the DMD gene and the HS6ST2 gene. The patient present with typical DMD symptoms such as muscle weakness, but no obvious symptoms of Paganini-Miozzo syndrome. Our results suggest that the destruction of DMD gene by structural rearrangement is also one of the important causes of DMD. Therefore, we suggest to provide further genetic testing for those DMD patients with unknown genetic defects through routine genetic testing. Cost-effective karyotyping and FISH should be considered firstly to identify chromosome rearrangements. Long-read sequencing followed by Sanger sequencing could be useful to locate the precise breakpoints. The genetic diagnosis of this case made it possible for reproductive intervention in the patient's family.

Biallelic CFAP61 variants cause male infertility in humans and mice with severe oligoasthenoteratozoospermia.

BACKGROUND: The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear. OBJECTIVE: To identify the genetic cause of male infertility characterised by OAT. METHODS: Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment. RESULTS: We identified biallelic CFAP61 variants (NM_015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61-deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby. CONCLUSIONS: Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT.

Novel MEIOB variants cause primary ovarian insufficiency and non-obstructive azoospermia.

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.

Pregnancy Outcomes of Different Endometrial Preparation in Patients With a History of Cesarean Section.

Objective: To investigate the efficacies of three cycle regimens in women receiving frozen embryo transfer with a history of cesarean section: natural cycle treatment, hormone replacement therapy and treatment with gonadotropin-releasing hormone agonist. Design: Retrospective cohort study. Methods: patients (N = 6,159) with a history of caesarean section who fulfilled the inclusion criteria were enrolled in the study from January 2014 to December 2019 at the CITIC-Xiangya Hospital of Reproduction and Genetics. Reproductive outcomes of patients in the natural cycle (n = 4,306) versus hormone replacement therapy (n = 1,007) versus gonadotropin-releasing hormone agonist + hormone replacement therapy groups (n = 846) were compared. Continuous data were analyzed using Student's t-test, and categorical variables were analyzed using the χ2 test. Multivariable logistic regression was used to evaluate the possible relationships between the types of endometrial preparation and pregnancy outcomes after adjusting for confounding factors. Results: The unadjusted odds of the miscarriage rate of singleton pregnancies were significantly higher in the hormone replacement therapy compared with the natural cycle (25.5% versus 20.4%, respectively). After adjusting for possible confounding factors, the early miscarriage rate and the miscarriage rate of singleton pregnancies remained significantly higher in the hormone replacement therapy than the natural cycle. The clinical pregnancy rates in the natural cycle, hormone replacement therapy and gonadotropin- releasing hormone agonist + hormone replacement therapy of women with a history of cesarean section was 48.8%, 48% and 47.1%, respectively, and the live birth rates were 37%, 34.1% and 35.7%, respectively. Conclusions: In women undergoing frozen embryo transfer with a history of cesarean section, hormone replacement therapy for endometrial preparation was associated with a higher early miscarriage rate, albeit after statistical adjustment for confounding factors. However, the risk observed was little and did not influence the overall reproductive performances.

Biallelic variants in MOS cause large polar body in oocyte and human female infertility.

STUDY QUESTION: What is the genetic basis of female infertility involving abnormal oocyte morphology with the production of a large first polar body (PB1)? SUMMARY ANSWER: The homozygous missense variant (c.791C>G) and compound missense variants (c.596A>T and c.875C>T) in MOS proto-oncogene, serine/threonine kinase (MOS) (Online Mendelian Inheritance in Man (OMIM) reference: 190060; NM_005372.1) are responsible for abnormal oocyte morphology with the production of a large PB1 to cause infertility in women. WHAT IS KNOWN ALREADY: MOS, an oocyte-specific gene, encodes a serine/threonine-protein kinase that directly phosphorylates mitogen-activated protein kinase (MAPK) kinase (MEK) to activate MAPK (also called extracellular-signal-regulated kinase (ERK)) signal cascade in the oocyte. Female mice lacking Mos remained viable, but infertile because of oocyte symmetric division, spontaneous parthenogenetic activation and early embryonic arrest. Recently, two independent studies demonstrated that female infertility with early embryonic arrest and fragmentation can be caused by biallelic mutations in MOS. However, so far, MOS variants have not been associated with the phenotype of large PB1 extrusion in human oocytes to contribute to female infertility. STUDY DESIGN, SIZE, DURATION: Two independent infertile families characterized by the presence of large PB1 in oocytes were recruited between December 2020 and February 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genomic DNA was extracted from the peripheral blood samples of the subjects for whole-exome sequencing. Pedigree analysis was validated by Sanger sequencing. Then, the pathogenic effects of the MOS variants on MOS protein properties and ERK1/2 activation were determined in HEK293 cells and mouse oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: We identified three rare missense variants in MOS, including a homozygous missense variant (c.791C>G) from Patient 1 in Family 1 and two compound missense variants (c.596A>T and c.875C>T) from twin sisters in Family 2. The MOS variants followed a recessive inheritance pattern in infertile patients. All three patients displayed a high percentage of large PB1 extrusion in the oocytes. The three MOS variants could not activate MEK1/2 and ERK1/2 in oocytes and HEK293 cells. In addition, when compared with wild-type MOS, the MOS variants decreased the MOS protein level and attenuated the binding capacity with MEK1. Microinjection of wild-type human MOS complementary RNAs (cRNAs) reversed the symmetric division of oocytes after siMos treatment. In contrast, the three MOS variants demonstrated no rescuing ability. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Owing to the scarcity of human oocyte samples and the associated ethical restrictions, we could not perform the rescue attempt for the study patients. WIDER IMPLICATIONS OF THE FINDINGS: Our findings expand the genetic and phenotypic spectrum of MOS variants in causing female infertility. Our study findings facilitate the early genetic diagnosis of abnormal oocyte morphology characterized as large PB1 that eventually causes infertility in women. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071640 and 82001633), Natural Science Foundation of Zhejiang Province (LD22C060001), the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project (WKJ-ZJ-2005), China Postdoctoral Science Foundation (2020M682575 and 2021T140198), the Changsha Municipal Natural Science Foundation (kq2007022) and Hunan Provincial Grant for Innovative Province Construction (2019SK4012). None of the authors declare any competing interests. TRIAL REGISTRATION NUMBER: N/A.

Trophectoderm Biopsy Differentially Influences the Level of Serum ß-Human Chorionic Gonadotropin With Different Embryonic Trophectoderm Scores in Early Pregnancy From 7847 Single-Blastocyst Transfer Cycles.

OBJECTIVE: To evaluate whether trophectoderm (TE) biopsy differentially influence the level of serum ß-human chorionic gonadotropin (ß-hCG) with different TE-scored blastocysts transferred in early pregnancy. METHODS: This retrospective cohort study contained 7847 single-blastocyst transfer cycles executed between January 2019 and June 2020, including 2657 preimplantation genetic testing (PGT) cycles and 5190 in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles. All cycles were classified into biopsy and control groups, and further stratified based on the TE morphological scores into three subgroups: grades A, B, and C for TE scores, respectively. Intra-group and inter-group analyses were performed on serum ß-hCG levels on the 12th day after blastocyst transfer (HCG12), and obstetric and neonatal outcomes. RESULTS: For cycles with a live birth, in grade A TE score subgroups, the HCG12 level did not exhibit statistical significance between the control and biopsy groups after adjustment (769 mIU/mL vs. 753 mIU/mL, P=0.631). In contrast, in grade B and C TE score subgroups, the control group showed a significantly higher level of HCG12 relative to the biopsy group (690 mIU/mL vs. 649 mIU/mL, P=0.001; 586 mIU/mL vs. 509 mIU/mL, P<0.001, respectively). We observed no statistically significant differences in obvious adverse obstetric and neonatal outcomes between the same TE-score subgroups of the biopsy group and control group. CONCLUSIONS: While blastocysts with higher TE grades produced higher serum ß-hCG levels in early pregnancy, TE biopsy might exert a negative impact on serum ß-hCG levels by blastocysts with a grade-B TE score and below. TE biopsy did not increase the risk for adverse obstetric and neonatal outcomes.

RNA splicing analysis contributes to reclassifying variants of uncertain significance and improves the diagnosis of monogenic disorders.

BACKGROUND: Numerous variants of uncertain significance (VUSs) have been identified by whole exome sequencing in clinical practice. However, VUSs are not currently considered medically actionable. OBJECTIVE: To assess the splicing patterns of 49 VUSs in 48 families identified clinically to improve genetic counselling and family planning. METHODS: Forty-nine participants with 49 VUSs were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Bioinformatic analysis was performed to preliminarily predict the splicing effects of these VUSs. RT-PCR and minigene analysis were used to assess the splicing patterns of the VUSs. According to the results obtained, couples opted for different methods of reproductive interventions to conceive a child, including prenatal diagnosis and preimplantation genetic testing (PGT). RESULTS: Eleven variants were found to alter pre-mRNA splicing and one variant caused nonsense-mediated mRNA decay, which resulted in the reclassification of these VUSs as likely pathogenic. One couple chose to undergo in vitro fertilisation with PGT treatment; a healthy embryo was transferred and the pregnancy is ongoing. Three couples opted for natural pregnancy with prenatal diagnosis. One couple terminated the pregnancy because the fetus was affected by short-rib thoracic dysplasia and harboured the related variant. The infants of the other two couples were born and were healthy at their last recorded follow-up. CONCLUSION: RNA splicing analysis is an important method to assess the impact of sequence variants on splicing in clinical practice and can contribute to the reclassification of a significant proportion of VUSs. RNA splicing analysis should be considered for genetic disease diagnostics.

Sperm flagellar 2 (SPEF2) is essential for sperm flagellar assembly in humans.

Spermiogenesis is a complex and tightly regulated process, consisting of acrosomal biogenesis, condensation of chromatin, flagellar assembly, and disposal of extra cytoplasm. Previous studies have reported that sperm flagellar 2 (SPEF2) deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure, but the underlying molecular mechanism in humans remains unclear. Here, we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development. A total of 1262 differentially expressed proteins were detected, including 486 upregulated and 776 downregulated. The constructed heat map of the differentially expressed proteins showed similar trends. Among these, the expression of proteins related to flagellar assembly, including SPEF2, sperm associated antigen 6 (SPAG6), dynein light chain tctex-type 1 (DYNLT1), radial spoke head component 1 (RSPH1), translocase of outer mitochondrial membrane 20 (TOM20), EF-hand domain containing 1 (EFHC1), meiosis-specific nuclear structural 1 (MNS1) and intraflagellar transport 20 (IFT20), was verified by western blot. Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly. Furthermore, we showed that SPEF2 interacts with radial spoke head component 9 (RSPH9) and IFT20 in vitro, which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly. These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.

Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation.

Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F-actin assembly. Using oocyte-specific Erk1/2 knockout mice, we verified that MOS-ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS-ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF.

CD19 and intraglandular CD163-positivity as prognostic indicators of pregnancy outcome in CD138-negative women with a previous fresh-embryo-transfer failure.

Many factors impede embryonic implantation, and excluding obvious known factors such as chronic endometritis, the immune status of the endometrium may be related to pregnancy. Although an abundantly large number of immune cells infiltrate the endometrium during the secretory phase, whether these immune cells can be used as a predictor of prognosis in ART has not yet been clarified. In the present study we therefore retrospectively analyzed 97 CD138-negative women with a previous fresh-embryo-transfer failure. We assessed the expression of CD56+ uNK cells, CD16+ NK cells, CD57+ NK cells, CD68+ pan-macrophages, CD163+ M2 macrophages, CD4+T cells, CD8+T cells, FOXP3+ regulatory T cells, and CD19+ B cells in the endometrium by IHC to evaluate mid-luteal endometrial immune cells as prognostic indicators of pregnancy outcome in the next frozen-embryo-transfer cycle. CD19-positive cells and the intraglandular CD163-positivity rate increased significantly in the clinically non-pregnant group (0.47 % vs. 0.20 %, P = 0.021; 61 % vs. 30 %, P = 0.017). The ratios of CD4/CD8 were also higher in the non-pregnant group (1.96 vs. 1.45, P = 0.005).The area under the ROC curve of CD19 cell number alone, the intraglandular CD163-positivity alone, and CD19 number combined with the intraglandular CD163-positivity were 0.692 (95 % CI, 0.55-0.834), 0.661 (95 % CI, 0.514-0.809), and 0.748 (95 % CI, 0.614-0.882), respectively. The optimal cut-off value of CD19 was 0.464 %, and the clinical pregnancy rate and live-birth rate diminished significantly when the CD19 level was above this cut-off value. Our study suggests that CD19-positive cells and intraglandular CD163-positivity can be used as prognostic indicators of pregnancy outcome in CD138-negative patients who experienced first-fresh-embryo transfer failure.

Next-generation sequence-based preimplantation genetic testing for monogenic disease resulting from maternal mosaicism.

BACKGROUND: Mosaicism poses challenges for genetic counseling and preimplantation genetic testing for monogenic disorders (PGT-M). NGS-based PGT-M has been extensively used to prevent the transmission of monogenic defects, but it has not been evaluated in the application of PGT-M resulting from mosaicism. METHODS: Four women suspected of mosaicism were confirmed by ultra-deep sequencing. Blastocyst trophectoderm cells and polar bodies were collected for whole genome amplification, followed by pathogenic variants detection and haplotype analysis based on NGS. The embryos free of the monogenic disorders were transplantable. RESULTS: Ultra-deep sequencing confirmed that the four women harbored somatic mosaic variants, with the proportion of variant cells at 1.12%, 9.0%, 27.60%, and 91.03%, respectively. A total of 25 blastocysts were biopsied and detected during four PGT cycles and 5 polar bodies were involved in one cycle additionally. For each couple, a wild-type embryo was successfully transplanted and confirmed by prenatal diagnosis, resulting in the birth of four healthy infants. CONCLUSIONS: Mosaic variants could be effectively evaluated via ultra-deep sequencing, and could be prevented the transmission by PGT. Our work suggested that an NGS-based PGT approach, involving pathogenic variants detection combined with haplotype analysis, is crucial for accurate PGT-M with mosaicism.