Novel LHX8 variants associated with distinctive oocyte morphological abnormalities and maturation arrest in primary infertility

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We evaluated two unrelated women with primary infertility who showed reproducible oocyte abnormalities across in vitro fertilization cycles, and we performed genomic and functional assays to clarify the role of LHX8 . Results Whole exome sequencing identified heterozygous loss-of-function variants in LHX8 (NM_001001933.1) in both patients: c.778C > T (p.Gln260Ter) in family 1 and c.581-1G > A in family 2. Both variants met the American College of Medical Genetics and Genomics criteria for likely pathogenicity. The two patients had high proportions of degenerated or immature oocytes and showed consistent morphologic features, including multiple cytoplasmic vacuoles, impaired zona pellucida function with accumulation of sperm in the perivitelline space, and poor embryo development. The splice site variant was inherited from a fertile mother, which indicates incomplete penetrance. A minigene assay confirmed the use of a cryptic acceptor site that produced a one nucleotide deletion and a frameshift, consistent with loss of function. Conclusions These findings expand the phenotypic spectrum of LHX8 related infertility and provide mechanistic evidence that partial reduction of LHX8 activity compromises oocyte quality. Recognition of the characteristic morphology may guide genetic testing and counseling in cases of unexplained infertility. LHX8 oocyte degeneration maturation arrest primary infertility Oocyte/Zygote/Embryo Maturation Arrest (OZEMA) Figures Figure 1 Figure 2 Figure 3 Background Infertility is a global health issue that will likely continue to rise through 2040. In 2021, it affected an estimated 110 million women and 55 million men worldwide, with a nearly two-fold higher prevalence in women than in men [ 1 ]. Within female infertility, oocyte maturation abnormalities (OMAS) and oocyte, zygote, or embryo maturation arrest (OZEMA) are increasingly recognized as important causes. Assisted reproductive technology (ART) makes it possible to observe oogenesis and early embryogenesis in detail and has refined these concepts. OMAS covers a spectrum of oocyte maturation defects, including degenerated or dysmorphic oocytes, empty follicle syndrome, oocyte maturation arrest (OMA), resistant ovary syndrome, and maturation disorders associated with primary ovarian insufficiency (POI) [ 2 ]. OZEMA refers to developmental arrest at the oocyte, zygote, or early embryo stage [ 3 ]. Successful oocyte maturation requires precise temporal and spatial control of gene networks that regulate meiotic progression, cytoskeletal dynamics, and organelle distribution. Disruption of these programs results in maturation failure and infertility. Genetic factors that influence these processes have attracted growing attention, and several infertility related genes have been identified, including TUBB8 and ZP1 [ 4 ]. TUBB8 variants cause oocyte maturation arrest through microtubule disruption, while ZP1 variants cause fertilization failure through defects in the zona pellucida. These insights have advanced our understanding of human gametogenesis and stimulated new approaches to diagnosis and counseling. LHX8 belongs to the LIM homeobox family and has a central role in embryonic development through transcriptional regulation of pattern formation and cell fate [ 5 ]. LIM homeodomain proteins contain two N terminal LIM domains that mediate protein interactions and a C terminal homeodomain that binds DNA in a sequence specific manner [ 6 ]. In mammals, LHX8 is expressed mainly in the ovary and functions as a germ cell specific transcription factor that is essential for oocyte differentiation and survival. In mouse models, deletion of Lhx8 leads to rapid oocyte loss and impaired follicle development from the primordial to the growing stages [ 7 ]. The human LHX8 gene on chromosome 1p31.1 has ten exons and is highly conserved across mammals, which indicates essential reproductive functions. Loss of function variants in LHX8 have recently been reported as a cause of oocyte maturation arrest and female infertility [ 8 ]. Although homozygous loss in mice causes severe reproductive abnormalities, heterozygous LHX8 variants may be sufficient to cause infertility in humans, which suggests species specific differences in gene dosage sensitivity. Reported cases show diverse phenotypes, including arrest at the germinal vesicle or metaphase I stage and morphological abnormalities in retrieved oocytes [ 8 ]. However, detailed morphological characterization in human heterozygous LHX8 variants remains limited. Here we report two women with primary infertility who carry novel LHX8 variants. Both showed distinctive oocyte morphology and developmental arrest. Our findings expand the phenotypic spectrum and provide new insight into the function of LHX8 in human reproduction. Methods Clinical samples Two patients with primary infertility from different families were recruited, each from a different fertility clinic. In Family 1, peripheral blood samples were collected from the proband (II-1) and her mother (I-2) (Fig. 1 A). In Family 2, samples were obtained from the proband (II-4), her mother (I-2), sister (II-1), and nephew (III-1) (Fig. 1 B). Comprehensive pedigree information was documented, including fertility history, age at conception, and any history of reproductive disorders. Genomic DNA was extracted from all samples for genetic testing and a segregation analysis. All participants provided written informed consent, and this study was approved by the Institutional Review Board of Fujita Health University (HG24-014). Clinical protocol and assisted reproductive procedures A controlled ovarian stimulation was started on day 3 of the menstrual cycle using one of three protocols: the gonadotropin-releasing hormone (GnRH) agonist protocol, GnRH antagonist protocol, or progestin-primed ovarian stimulation (PPOS). Protocol selection was based on each subject’s characteristics, including age, anti-Müllerian hormone (AMH) levels, and previous ovarian responses. In the GnRH agonist protocol, patients received buserelin acetate (Suprecur; Mochida Pharmaceutical, Tokyo, Japan) daily starting from day 3 of the menstrual cycle and continuing throughout the stimulation period. In the GnRH antagonist protocol, cetrorelix acetate (Cetrotide; Merck Biopharma, Tokyo, Japan) was administered when the leading follicle reached 14 mm in diameter. In PPOS, dydrogesterone (Duphaston; Mylan EPD, Tokyo, Japan) was administered from cycle day 3 until the trigger day. Follicular development was monitored by transvaginal ultrasonography and serum estradiol measurements. When the two leading follicles reached at least 20 mm in diameter, human chorionic gonadotropin (hCG; Mochida Pharmaceutical, Tokyo, Japan) 5000 IU was administered. Oocyte retrieval was performed 36–38 hours after the hCG trigger under transvaginal ultrasound guidance with sedation. Retrieved oocytes were inseminated by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection based on semen parameters or fertilization rates. The fertilization status was assessed 16–18 hours post-insemination by checking for the presence of two pronuclei. Whole-exome sequencing (WES) and a variant analysis Genomic DNA was extracted from 400 µL of peripheral blood or saliva samples using the magLEAD system (Precision System Science, Chiba, Japan). WES was performed on the NovaSeq 6000 platform (Illumina, San Diego, CA). Raw sequence data (FASTQ files) were aligned to the human reference genome (UCSC hg38/GRCh38). A coverage analysis revealed that at least 97% of exons had > 30× coverage. Variant calling and filtering were conducted based on quality metrics, minor allele frequency (MAF), and the predicted functional impact. Allele frequencies were obtained from the Genome Aggregation Database (gnomAD v4.1.0; https://gnomad.broadinstitute.org/ ) and the ToMMo 8.3KJPNv2 database (Tohoku Medical Megabank Organization; 8,380 Japanese individuals, https://jmorp.megabank.tohoku.ac.jp/help/tutorial ) using an MAF cut-off of < 0.01 in both cases. Candidate variants identified by WES were validated by Sanger sequencing. PCR primers flanking the regions of interest were designed using Primer3 software ( https://bioinfo.ut.ee/primer3/ ). After amplification, PCR products were purified and sequenced using the SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA). The pathogenicity of validated variants was classified according to the 2015 ACMG/AMP guidelines (American College of Medical Genetics and Genomics / Association for Molecular Pathology). Computational tools, such as gnomAD metrics (probability of being loss-of-function intolerant (pLI) and loss-of-function observed/expected upper bound fraction (LOEUF) scores; https://gnomad.broadinstitute.org/ ), DECIPHER prediction tools (probability of haploinsufficiency (pHaplo), probability of triplosensitivity, and probability of loss-of-function mechanism (pLOF) scores) https://www.deciphergenomics.org/ ), and SpliceAI ( https://spliceailookup.broadinstitute.org/ ), were used to predict the functional impact of the variants identified. Minigene analysis A minigene splicing assay was performed as previously described [ 9 ]. Briefly, the LHX8 genomic region encompassing exon 6, intron 6, and exon 7 with flanking sequences was amplified from patient and control genomic DNA using primers containing XhoI and SpeI restriction sites. The resulting PCR products were subcloned into the multiple cloning site of the pET01 Exontrap vector (MoBiTec). These plasmids were transfected into HEK293 cells using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were harvested 48 hours after transfection, and total RNA was isolated using the RNeasy Mini Kit (Qiagen). First-strand cDNA was synthesized using SuperScript III (Invitrogen) with oligo(dT) primers. RT-PCR was performed with vector-specific primers, and PCR products were analyzed by 2% agarose gel electrophoresis and direct Sanger sequencing to evaluate splicing patterns. Results Clinical Phenotypes The clinical characteristics of the study patients are summarized in Table 1 . In Family 1, the patient (II-1) was a 31-year-old female who presented with a 5-year history of infertility. She had been diagnosed with unexplained infertility and had undergone three months of timed intercourse followed by 12 cycles of intrauterine insemination; however, pregnancy was not achieved. Consequently, the decision was made to proceed with ART. The patient had a normal ovarian reserve with an AMH level of 2.02 ng/mL. All parameters (concentration, motility, and morphology) were within normal ranges in her spouse’s semen. Table 1 Clinical characteristics of study patients Age (years) AMH (ng/mL) Duration of infertility (years) Number of retrievals Total number of oocytes GV oocyte (n) MI oocyte (n) MII oocyte (n) Oocytes with an abnormal morphology (n) Fertilized oocytes (n) Viable embryos (n) Features Family 1 (Ⅱ-1) 31 2.02 5 6 126 15 29 54 21 51 1 Frequent cytoplasmic vacuoles and polyspermy in PVS Family 2 (Ⅱ-4) 27 1.62 4 7 33 2 0 15 14 5 0 Mainly polyspermy in PVS Abbreviations: GV, Germinal Vesicle; MI, Metaphase I; MII, Metaphase II; PVS, perivitelline space. Six oocyte retrieval cycles were performed, during which 126 oocytes were retrieved (15 GV, 29 MI, and 54 metaphase II (MII) oocytes). Among these oocytes, 21 (16.7%) had an abnormal morphology characterized by an abnormal zona pellucida with partial thinning (Fig. 2 a), multiple cytoplasmic vacuoles (Fig. 2 b), and multiple spermatozoa penetration into the perivitelline space after conventional IVF (Fig. 2 c). In fertilized oocytes, cytoplasmic vacuoles increased during the pronuclear stage. Despite multiple attempts, with 51 oocytes being successfully fertilized, only one viable embryo developed to a day 6 blastocyst with Gardner grade 3BC. This blastocyst was transferred, but did not result in pregnancy. In Family 2, the patient (II-4) was a 27-year-old female with a 4-year history of infertility and an AMH level of 1.62 ng/mL. The spousal semen analysis revealed that all parameters were within normal ranges. Thirty-three oocytes were retrieved (2 GV, 0 MI, and 15 MII oocytes) from seven cycles. Similar to Family 1, many oocytes (14/33, 42.4%) had an abnormal morphology. These abnormalities were characterized predominantly by multiple sperm penetration into the perivitelline space following conventional IVF (Fig. 2 d, e) and cytoplasmic vacuoles at the pronuclear stage (Fig. 2 f). Five oocytes achieved fertilization; however, no viable embryos were obtained. The patient’s mother (Family2, I-2) with the same variant had given birth to four children at the ages of 22, 24, 30, and 32 years. She had no history of infertility treatment and experienced menopause at approximately 50 years of age. The patient’s sister (II-1) had conceived naturally at the age of 28 years without any infertility issues. Her other siblings (II-2 and II-3) were unmarried at the time of the analysis. Variant Analysis A genetic analysis identified heterozygous LHX8 variants in both probands (Table 2 ). In Family 1, the proband (II-1) carried the nonsense variant c.778C > T, which was not found in her mother (I-2). Since a paternal sample was unavailable, this variant indicated de novo or paternal inheritance (Fig. 1 A). In Family 2, the proband (II-4) carried the heterozygous splice site variant c.581-1G > A, which was inherited from her mother (I-2). Sanger sequencing confirmed that the proband’s sister (II-1) and her son (III-1) were both negative for this variant (Fig. 1 B). Table 2 Overview of LHX8 variants Variants Variant type Zygosity gnomAD ToMMo ACMG criteria ACMG pathogenicity class Family 1 (Ⅱ-1) c.778C > T(p.Q260*) Nonsense Heterozygous NA NA PVS1, PM2 Likely Pathogenic Family 2 (Ⅱ-4) c.581-1G > A Splicing Heterozygous NA NA PVS1, PM2 Likely Pathogenic NA, not available The nonsense variant c.778C > T (p.Gln260Ter) in Family 1 introduced a premature termination codon in exon 7. Regarding the splice site variant c.581-1G > A in Family 2, a sequence analysis confirmed that it affected the invariant G nucleotide at the − 1 position of the canonical splice acceptor site. This nucleotide position showed complete conservation across vertebrate species in the LHX8 gene. Both variants were absent from population databases (gnomAD and ToMMo), indicating that they are not common polymorphisms in the general population. To examine the pathogenicity of the LHX8 variants in the present study, we utilized complementary metrics from multiple databases. LHX8 demonstrated an intolerance to loss-of-function variants, with gnomAD pLI of 0.97 and LOEUF of 0.52. A DECIPHER analysis indicated pHaplo of 0.75 and pLOF of 0.655. Regarding the splice site variant c.581-1G > A, SpliceAI predicted high probabilities of acceptor loss (0.99) and splice loss (0.85). Additionally, this variant showed a significant acceptor gain score (0.94) 2 bp from the original site, suggesting the activation of a cryptic splice site. According to the ACMG/AMP guidelines, both variants were classified as likely pathogenic, fulfilling criteria PVS1 and PM2 in both cases (Table 2 ). Functional Analysis of the c.581-1G > A Splice Site Variant To investigate the functional consequences of the c.581-1G > A variant on LHX8 splicing, we performed minigene splicing assays using the Exontrap system. The genomic fragment containing LHX8 exons 6 and 7 with the intervening intron 6 was cloned into the pET01 vector and transfected into HEK293 cells. The RT-PCR analysis of transfected cells revealed distinct splicing patterns between the wild-type (WT) and patient (PT) constructs. The WT minigene produced the expected transcript containing exons 6 and 7, with the normal splicing of intron 6 (Fig. 3 A, lane 2). No clear differences were detected in PCR product sizes between WT and PT constructs by agarose gel electrophoresis (Fig. 3 B). Sanger sequencing of RT-PCR products revealed that the c.581-1G > A variant resulted in the use of an aberrant splice site located 1 bp downstream of the original acceptor site (Fig. 3 C-D). This led to a 1-bp deletion in the mature transcript from the WT sequence. This splicing pattern was consistently observed across three independent experiments and aligned with SpliceAI predictions. Discussion We identified two women with primary infertility who carry novel LHX8 variants, each with high percentages of degenerated and immature oocytes. Despite different variants, the cases showed two common features: accumulation of sperm within the perivitelline space and increased cytoplasmic vacuoles in oocytes at the pronuclear stage. These morphological abnormalities may be understood in the context of the function of LHX8 . LHX8 acts as a key transcription factor in early oogenesis and interacts with SOHLH1 to form a nuclear complex [ 7 , 10 ]. This complex coordinates the expression of downstream genes that are essential for oocyte growth and differentiation, including Zp1 and Zp3 [ 11 ]. Zona pellucida proteins form the glycoprotein matrix around the oocyte and have central roles in species specific sperm recognition, binding, and prevention of polyspermy [ 12 ]. The observed sperm accumulation suggests zona pellucida dysfunction, which may reflect altered expression or function of these proteins. Cytoplasmic vacuoles are established markers of reduced oocyte competence and are associated with lower fertilization rates and poorer outcomes [ 13 ]. Collectively, these abnormalities support the view that LHX8 regulates oocyte quality through multiple pathways. Further functional studies are needed to clarify the diverse effects of LHX8 variants in oogenesis. Our results extend previous reports on LHX8 related infertility. Zhao et al. described heterozygous loss of function LHX8 variants, including splicing, nonsense, and frameshift variants, caused human infertility [ 8 ]. The type of variant may modify the phenotype, as missense variants have been reported in patients with POI [ 14 ], and common polymorphisms have been associated with the risk of POI in genome-wide studies [ 15 ]. In our study, both patients carried null variants and had normal ovarian reserve, with infertility as the only clinical finding. The minigene assay confirmed the use of a cryptic splice acceptor site 2 bp downstream, resulting in a 1 bp deletion and frameshift as predicted by SpliceAI. Despite this functional evidence, incomplete penetrance was evident in family 2, where the mother carried the same variant and was fertile. The pedigree, in which the proband's sister and her child did not inherit the variant and had normal fertility, supports incomplete penetrance. This has practical implications for counseling because clinical expression can be modified by trans-acting factors [ 16 , 17 ], epigenetic changes, and environmental factors such as age and hormonal prolife. These observations also support interspecies differences in dosage sensitivity. While homozygous deletion of Lhx8 results in a phenotype in mice, heterozygous LHX8 variants appear to be sufficient to cause reproductive abnormalities in humans [ 8 , 18 ]. This pattern, together with high constraint metrics such as a pLI of 0.97 and a pHaplo of 0.75 in our cases, suggests that human oogenesis is sensitive to LHX8 dosage. At the cellular level, recent work in mice links Lhx8 deficiency to altered autophagy [ 19 ]. The vacuoles we observed may reflect a related disruption in cellular quality control. Clinically, our data suggest that LHX8 testing may be considered for women with recurrent OMA, particularly when cytoplasmic vacuoles or abnormal perivitelline sperm accumulation are present. Conclusion We report two clinical cases with novel LHX8 variants (c.778C > T and c.581-1G > A) that are likely pathogenic according to ACMG and AMP guidelines. The consistent morphological and developmental findings support the central role of LHX8 in oocyte maturation. These features may guide genetic testing and counseling for unexplained infertility. Declarations Ethics approval Ethical approval was obtained from the Institutional Review Board of the Fujita Health University (HG24-014). Written informed consent was obtained from both participants prior to their inclusion in this study. Competing interests The authors declare no competing interests in relation to this study. Funding This research was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP25ek0109760h0002. Author Contribution All authors participated in the study’s design and revisions to the manuscript. Each author approved the final version and takes responsibility for all aspects of the work. Specifically, YS, HI, and HK conceptualized and designed the study. RY and HN provided critical feedback and helped in shaping the research, analysis, and manuscript. KK, YM, and TH contributed to the interpretation of clinical data. KN, KY, and RH performed laboratory experiments and data collection. HN and HK supervised the project, provided expertise for data interpretation, and secured funding. YS, HI, and HK drafted the initial manuscript and created the tables and figures. Data Availability The datasets generated and/or analyzed during the present study are available from the corresponding author upon reasonable request. References Liang Y, Huang J, Zhao Q, Mo H, Su Z, Feng S, et al. Global, regional, and national prevalence and trends of infertility among individuals of reproductive age (15–49 years) from 1990 to 2021, with projections to 2040. Hum Reprod. 2025;40:529–44. Hatırnaz Ş, Hatırnaz ES, Ellibeş Kaya A, Hatırnaz K, Soyer Çalışkan C, Sezer Ö, et al. Oocyte maturation abnormalities - A systematic review of the evidence and mechanisms in a rare but difficult to manage fertility pheneomina. Turk J Obstet Gynecol. 2022;19:60–80. Van Der Kelen A, Uyttebroeck S, Van de Voorde S, Picchetta L, Segers I, Vlaeminck J, et al. Oocyte/zygote/embryo maturation arrest: a clinical study expanding the phenotype of NOBOX variants. J Assist Reprod Genet. 2025;42:763–71. Feng R, Sang Q, Kuang Y, Sun X, Yan Z, Zhang S, et al. Mutations in TUBB8 and Human Oocyte Meiotic Arrest. N Engl J Med. 2016;374:223–32. Kitanaka J, Takemura M, Matsumoto K, Mori T, Wanaka A. Structure and chromosomal localization of a murine LIM/homeobox gene, Lhx8. Genomics. 1998;49:307–9. Fu L, Zhang M, Mastrantoni K, Perfetto M, Wei S, Yao J. Bovine Lhx8, a germ cell-specific nuclear factor, interacts with Figla. PLoS ONE. 2016;11:e0164671. Choi Y, Ballow DJ, Xin Y, Rajkovic A. Lim homeobox gene, lhx8, is essential for mouse oocyte differentiation and survival. Biol Reprod. 2008;79:442–9. Zhao L, Li Q, Kuang Y, Xu P, Sun X, Meng Q, et al. Heterozygous loss-of-function variants in LHX8 cause female infertility characterized by oocyte maturation arrest. Genet Med. 2022;24:2274–84. Bolor H, Mori T, Nishiyama S, Ito Y, Hosoba E, Inagaki H, et al. Mutations of the SYCP3 gene in women with recurrent pregnancy loss. Am J Hum Genet. 2009;84:14–20. Wang Z, Liu C-Y, Zhao Y, Dean J, FIGLA. LHX8 and SOHLH1 transcription factor networks regulate mouse oocyte growth and differentiation. Nucleic Acids Res. 2020;48:3525–41. Pangas SA, Choi Y, Ballow DJ, Zhao Y, Westphal H, Matzuk MM, et al. Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8. Proc Natl Acad Sci U S A. 2006;103:8090–5. Wassarman PM. Zona pellucida glycoproteins. J Biol Chem. 2008;283:24285–9. Nikiforov D, Grøndahl ML, Hreinsson J, Andersen CY. Human oocyte morphology and outcomes of infertility treatment: A systematic review. Reprod Sci. 2022;29:2768–85. Bouilly J, Beau I, Barraud S, Bernard V, Azibi K, Fagart J, et al. Identification of multiple gene mutations accounts for a new genetic architecture of primary ovarian insufficiency. J Clin Endocrinol Metab. 2016;101:4541–50. Qin Y, Zhao H, Kovanci E, Simpson JL, Chen Z-J, Rajkovic A. Analysis of LHX8 mutation in premature ovarian failure. Fertil Steril. 2008;89:1012–4. Cooper DN, Krawczak M, Polychronakos C, Tyler-Smith C, Kehrer-Sawatzki H. Where genotype is not predictive of phenotype: towards an understanding of the molecular basis of reduced penetrance in human inherited disease. Hum Genet. 2013;132:1077–130. Castel SE, Cervera A, Mohammadi P, Aguet F, Reverter F, Wolman A, et al. Modified penetrance of coding variants by cis-regulatory variation contributes to disease risk. Nat Genet. 2018;50:1327–34. Ren Y, Suzuki H, Jagarlamudi K, Golnoski K, McGuire M, Lopes R, et al. Lhx8 regulates primordial follicle activation and postnatal folliculogenesis. BMC Biol. 2015;13:39. D’Ignazio L, Michel M, Beyer M, Thompson K, Forabosco A, Schlessinger D, et al. Lhx8 ablation leads to massive autophagy of mouse oocytes associated with DNA damage. Biol Reprod. 2018;98:532–42. Additional Declarations No competing interests reported. Supplementary Files supplementaryFigureS1.tif Supplementary Figure S1. Full uncropped agarose gel images corresponding to Fig. 3B, including all lanes and DNA size markers. Cite Share Download PDF Status: Published Journal Publication published 21 Jan, 2026 Read the published version in Journal of Ovarian Research → Version 1 posted Editorial decision: Revision requested 06 Nov, 2025 Reviews received at journal 05 Nov, 2025 Reviews received at journal 03 Nov, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers invited by journal 07 Oct, 2025 Editor assigned by journal 08 Sep, 2025 Submission checks completed at journal 08 Sep, 2025 First submitted to journal 26 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7460153","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":531421288,"identity":"4012e3be-f456-463b-9952-4728a1c40557","order_by":0,"name":"Yusuke Sako","email":"","orcid":"","institution":"Fujita Health University","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Sako","suffix":""},{"id":531421290,"identity":"cb7174ec-5729-422c-9467-852033ec4464","order_by":1,"name":"Hidehito Inagaki","email":"","orcid":"","institution":"Fujita Health 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07:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7460153/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7460153/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-026-01978-2","type":"published","date":"2026-01-21T15:58:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93916001,"identity":"9e02724b-c85a-4cd4-9d93-badb45eb15c6","added_by":"auto","created_at":"2025-10-20 08:51:11","extension":"tiff","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26007182,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/4a49f77bcb8834585d36a4de.tiff"},{"id":93915154,"identity":"b10f5289-050d-4e31-945f-e104ba978a76","added_by":"auto","created_at":"2025-10-20 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08:43:11","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85042,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/63dd741810f7279767f639fe.html"},{"id":93915998,"identity":"31032fbc-83af-4365-9c2e-392ec06d83dc","added_by":"auto","created_at":"2025-10-20 08:51:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148255,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of\u003cem\u003e LHX8\u003c/em\u003e variants in two families.\u003c/p\u003e\n\u003cp\u003eA. Pedigree of family 1 and Sanger sequencing chromatograms showing the heterozygous LHX8 nonsense variant c.778C\u0026gt;T (p.Gln260Ter) in the female proband (II-1). The variant was not detected in the proband's mother (I-2).\u003c/p\u003e\n\u003cp\u003eB. Pedigree of family 2 and Sanger sequencing chromatograms showing the heterozygous LHX8 splice site variant c.581-1G\u0026gt;A in the female proband (II-4) and her mother (I-2). This variant was not detected in the father (I-1), the proband's sister (II-1), or her nephew (III-1), as indicated by wild-type sequences identical to the control.\u003c/p\u003e\n\u003cp\u003eC. Schematic representation of the LHX8 gene structure and protein domains showing the location of the identified variants. Arrows indicate the positions of the variants.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/8e51f60e8cf1f00f018f0a29.png"},{"id":93915155,"identity":"e1426a46-3294-461a-ba07-4ac71471f551","added_by":"auto","created_at":"2025-10-20 08:43:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":402500,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological abnormalities in proband oocytes during fertilization.\u003c/p\u003e\n\u003cp\u003eRepresentative images of oocytes collected from family 1 proband (II-1, a-c) and family 2 proband (II-4, d-f). (a) Oocyte with abnormal zona pellucida with partial thinning and degenerative changes (white arrows). (b) Oocyte from family 1 proband with multiple cytoplasmic vacuoles. (c) Oocyte with multiple spermatozoa in the perivitelline space (yellow arrows). (d, e) Oocytes from family 2 proband with multiple spermatozoa in the perivitelline space (yellow arrows). (f) Oocyte from family 2 proband with multiple cytoplasmic vacuoles.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/251998eb60bcb92593bab762.png"},{"id":93915157,"identity":"3ecc15e1-fa27-4b38-8afc-dd2dab64636b","added_by":"auto","created_at":"2025-10-20 08:43:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71721,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of the \u003cem\u003eLHX8\u003c/em\u003ec.581-1G\u0026gt;A splice site variant using minigene assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the minigene construct and RT-PCR analysis. The \u003cem\u003eLHX8 \u003c/em\u003egenomic fragment containing exons 6 and 7 with intervening intron 6 was cloned into the pET01 Exontrap vector. Expected PCR product sizes are shown for the WT (595 bp) and PT (594 bp) constructs. Dotted lines indicate splicing events; solid lines indicate retained sequences. *Asterisk indicates the variant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e 2% agarose gel electrophoresis of RT-PCR products from HEK293 cells transfected with WT and PT constructs. Lane M, molecular weight marker; lane 1, WT construct; lane 2, PT construct; lane 3, mock transfection control (NC). GAPDH was used as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Sanger sequencing chromatograms showing the splice junction region. Upper panel: WT sequence showing normal splicing between exons 6 and 7. Lower panel: PT sequence showing aberrant splicing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Schematic illustration of the splicing pattern. The c.581-1G\u0026gt;A variant abolishes the canonical splice acceptor site, resulting in the activation of a cryptic splice site located 2 bp downstream. This results in a 1-bp deletion in the mature transcript from the WT sequence. Dotted lines indicate the splicing events.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/6477215d96b0ac5ccdd1eb79.png"},{"id":101151847,"identity":"4a41de77-4e2d-409e-b651-0c1a474dec0c","added_by":"auto","created_at":"2026-01-26 16:06:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1556972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/a784fe8f-47f0-4241-a547-da6c99bbe213.pdf"},{"id":93915160,"identity":"b60fe2d2-c301-42e4-a247-530dbe270cd5","added_by":"auto","created_at":"2025-10-20 08:43:11","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":163386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1. \u003c/strong\u003eFull uncropped agarose gel images corresponding to Fig. 3B, including all lanes and DNA size markers.\u003c/p\u003e","description":"","filename":"supplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7460153/v1/654328202857a312d61c6da9.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel LHX8 variants associated with distinctive oocyte morphological abnormalities and maturation arrest in primary infertility","fulltext":[{"header":"Background","content":"\u003cp\u003eInfertility is a global health issue that will likely continue to rise through 2040. In 2021, it affected an estimated 110\u0026nbsp;million women and 55\u0026nbsp;million men worldwide, with a nearly two-fold higher prevalence in women than in men [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Within female infertility, oocyte maturation abnormalities (OMAS) and oocyte, zygote, or embryo maturation arrest (OZEMA) are increasingly recognized as important causes. Assisted reproductive technology (ART) makes it possible to observe oogenesis and early embryogenesis in detail and has refined these concepts. OMAS covers a spectrum of oocyte maturation defects, including degenerated or dysmorphic oocytes, empty follicle syndrome, oocyte maturation arrest (OMA), resistant ovary syndrome, and maturation disorders associated with primary ovarian insufficiency (POI) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. OZEMA refers to developmental arrest at the oocyte, zygote, or early embryo stage [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Successful oocyte maturation requires precise temporal and spatial control of gene networks that regulate meiotic progression, cytoskeletal dynamics, and organelle distribution. Disruption of these programs results in maturation failure and infertility. Genetic factors that influence these processes have attracted growing attention, and several infertility related genes have been identified, including \u003cem\u003eTUBB8\u003c/em\u003e and \u003cem\u003eZP1\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eTUBB8\u003c/em\u003e variants cause oocyte maturation arrest through microtubule disruption, while \u003cem\u003eZP1\u003c/em\u003e variants cause fertilization failure through defects in the zona pellucida. These insights have advanced our understanding of human gametogenesis and stimulated new approaches to diagnosis and counseling.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLHX8\u003c/em\u003e belongs to the LIM homeobox family and has a central role in embryonic development through transcriptional regulation of pattern formation and cell fate [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. LIM homeodomain proteins contain two N terminal LIM domains that mediate protein interactions and a C terminal homeodomain that binds DNA in a sequence specific manner [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In mammals, \u003cem\u003eLHX8\u003c/em\u003e is expressed mainly in the ovary and functions as a germ cell specific transcription factor that is essential for oocyte differentiation and survival. In mouse models, deletion of \u003cem\u003eLhx8\u003c/em\u003e leads to rapid oocyte loss and impaired follicle development from the primordial to the growing stages [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The human \u003cem\u003eLHX8\u003c/em\u003e gene on chromosome 1p31.1 has ten exons and is highly conserved across mammals, which indicates essential reproductive functions.\u003c/p\u003e\u003cp\u003eLoss of function variants in \u003cem\u003eLHX8\u003c/em\u003e have recently been reported as a cause of oocyte maturation arrest and female infertility [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although homozygous loss in mice causes severe reproductive abnormalities, heterozygous \u003cem\u003eLHX8\u003c/em\u003e variants may be sufficient to cause infertility in humans, which suggests species specific differences in gene dosage sensitivity. Reported cases show diverse phenotypes, including arrest at the germinal vesicle or metaphase I stage and morphological abnormalities in retrieved oocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, detailed morphological characterization in human heterozygous \u003cem\u003eLHX8\u003c/em\u003e variants remains limited.\u003c/p\u003e\u003cp\u003eHere we report two women with primary infertility who carry novel \u003cem\u003eLHX8\u003c/em\u003e variants. Both showed distinctive oocyte morphology and developmental arrest. Our findings expand the phenotypic spectrum and provide new insight into the function of \u003cem\u003eLHX8\u003c/em\u003e in human reproduction.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eClinical samples\u003c/h2\u003e\u003cp\u003eTwo patients with primary infertility from different families were recruited, each from a different fertility clinic. In Family 1, peripheral blood samples were collected from the proband (II-1) and her mother (I-2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In Family 2, samples were obtained from the proband (II-4), her mother (I-2), sister (II-1), and nephew (III-1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Comprehensive pedigree information was documented, including fertility history, age at conception, and any history of reproductive disorders. Genomic DNA was extracted from all samples for genetic testing and a segregation analysis. All participants provided written informed consent, and this study was approved by the Institutional Review Board of Fujita Health University (HG24-014).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eClinical protocol and assisted reproductive procedures\u003c/h3\u003e\n\u003cp\u003eA controlled ovarian stimulation was started on day 3 of the menstrual cycle using one of three protocols: the gonadotropin-releasing hormone (GnRH) agonist protocol, GnRH antagonist protocol, or progestin-primed ovarian stimulation (PPOS). Protocol selection was based on each subject\u0026rsquo;s characteristics, including age, anti-M\u0026uuml;llerian hormone (AMH) levels, and previous ovarian responses. In the GnRH agonist protocol, patients received buserelin acetate (Suprecur; Mochida Pharmaceutical, Tokyo, Japan) daily starting from day 3 of the menstrual cycle and continuing throughout the stimulation period. In the GnRH antagonist protocol, cetrorelix acetate (Cetrotide; Merck Biopharma, Tokyo, Japan) was administered when the leading follicle reached 14 mm in diameter. In PPOS, dydrogesterone (Duphaston; Mylan EPD, Tokyo, Japan) was administered from cycle day 3 until the trigger day. Follicular development was monitored by transvaginal ultrasonography and serum estradiol measurements. When the two leading follicles reached at least 20 mm in diameter, human chorionic gonadotropin (hCG; Mochida Pharmaceutical, Tokyo, Japan) 5000 IU was administered. Oocyte retrieval was performed 36\u0026ndash;38 hours after the hCG trigger under transvaginal ultrasound guidance with sedation. Retrieved oocytes were inseminated by conventional \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) or intracytoplasmic sperm injection based on semen parameters or fertilization rates. The fertilization status was assessed 16\u0026ndash;18 hours post-insemination by checking for the presence of two pronuclei.\u003c/p\u003e\n\u003ch3\u003eWhole-exome sequencing (WES) and a variant analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from 400 \u0026micro;L of peripheral blood or saliva samples using the magLEAD system (Precision System Science, Chiba, Japan). WES was performed on the NovaSeq 6000 platform (Illumina, San Diego, CA). Raw sequence data (FASTQ files) were aligned to the human reference genome (UCSC hg38/GRCh38). A coverage analysis revealed that at least 97% of exons had\u0026thinsp;\u0026gt;\u0026thinsp;30\u0026times; coverage. Variant calling and filtering were conducted based on quality metrics, minor allele frequency (MAF), and the predicted functional impact. Allele frequencies were obtained from the Genome Aggregation Database (gnomAD v4.1.0; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://gnomad.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the ToMMo 8.3KJPNv2 database (Tohoku Medical Megabank Organization; 8,380 Japanese individuals, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jmorp.megabank.tohoku.ac.jp/help/tutorial\u003c/span\u003e\u003cspan address=\"https://jmorp.megabank.tohoku.ac.jp/help/tutorial\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using an MAF cut-off of \u0026lt;\u0026thinsp;0.01 in both cases.\u003c/p\u003e\u003cp\u003eCandidate variants identified by WES were validated by Sanger sequencing. PCR primers flanking the regions of interest were designed using Primer3 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfo.ut.ee/primer3/\u003c/span\u003e\u003cspan address=\"https://bioinfo.ut.ee/primer3/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). After amplification, PCR products were purified and sequenced using the SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA). The pathogenicity of validated variants was classified according to the 2015 ACMG/AMP guidelines (American College of Medical Genetics and Genomics / Association for Molecular Pathology). Computational tools, such as gnomAD metrics (probability of being loss-of-function intolerant (pLI) and loss-of-function observed/expected upper bound fraction (LOEUF) scores; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://gnomad.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), DECIPHER prediction tools (probability of haploinsufficiency (pHaplo), probability of triplosensitivity, and probability of loss-of-function mechanism (pLOF) scores) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.deciphergenomics.org/\u003c/span\u003e\u003cspan address=\"https://www.deciphergenomics.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and SpliceAI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://spliceailookup.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://spliceailookup.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), were used to predict the functional impact of the variants identified.\u003c/p\u003e\n\u003ch3\u003eMinigene analysis\u003c/h3\u003e\n\u003cp\u003eA minigene splicing assay was performed as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Briefly, the \u003cem\u003eLHX8\u003c/em\u003e genomic region encompassing exon 6, intron 6, and exon 7 with flanking sequences was amplified from patient and control genomic DNA using primers containing XhoI and SpeI restriction sites. The resulting PCR products were subcloned into the multiple cloning site of the pET01 Exontrap vector (MoBiTec). These plasmids were transfected into HEK293 cells using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were harvested 48 hours after transfection, and total RNA was isolated using the RNeasy Mini Kit (Qiagen). First-strand cDNA was synthesized using SuperScript III (Invitrogen) with oligo(dT) primers. RT-PCR was performed with vector-specific primers, and PCR products were analyzed by 2% agarose gel electrophoresis and direct Sanger sequencing to evaluate splicing patterns.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eClinical Phenotypes\u003c/h2\u003e\u003cp\u003eThe clinical characteristics of the study patients are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In Family 1, the patient (II-1) was a 31-year-old female who presented with a 5-year history of infertility. She had been diagnosed with unexplained infertility and had undergone three months of timed intercourse followed by 12 cycles of intrauterine insemination; however, pregnancy was not achieved. Consequently, the decision was made to proceed with ART. The patient had a normal ovarian reserve with an AMH level of 2.02 ng/mL. All parameters (concentration, motility, and morphology) were within normal ranges in her spouse\u0026rsquo;s semen.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eClinical characteristics of study patients\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eAge\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(years)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eAMH\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(ng/mL)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDuration of infertility\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(years)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eNumber of retrievals\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eTotal number of oocytes\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eGV\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eoocyte\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(n)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eMI\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eoocyte\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(n)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eMII\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eoocyte\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(n)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003eOocytes with an abnormal\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003emorphology\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(n)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eFertilized oocytes\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(n)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cem\u003eViable\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eembryos\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e(n)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cem\u003eFeatures\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily 1\u003c/p\u003e\u003cp\u003e(Ⅱ-1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eFrequent cytoplasmic vacuoles and polyspermy in PVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily 2\u003c/p\u003e\u003cp\u003e(Ⅱ-4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eMainly polyspermy in PVS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"13\"\u003eAbbreviations: GV, Germinal Vesicle; MI, Metaphase I; MII, Metaphase II; PVS, perivitelline space.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSix oocyte retrieval cycles were performed, during which 126 oocytes were retrieved (15 GV, 29 MI, and 54 metaphase II (MII) oocytes). Among these oocytes, 21 (16.7%) had an abnormal morphology characterized by an abnormal zona pellucida with partial thinning (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), multiple cytoplasmic vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and multiple spermatozoa penetration into the perivitelline space after conventional IVF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In fertilized oocytes, cytoplasmic vacuoles increased during the pronuclear stage. Despite multiple attempts, with 51 oocytes being successfully fertilized, only one viable embryo developed to a day 6 blastocyst with Gardner grade 3BC. This blastocyst was transferred, but did not result in pregnancy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn Family 2, the patient (II-4) was a 27-year-old female with a 4-year history of infertility and an AMH level of 1.62 ng/mL. The spousal semen analysis revealed that all parameters were within normal ranges. Thirty-three oocytes were retrieved (2 GV, 0 MI, and 15 MII oocytes) from seven cycles. Similar to Family 1, many oocytes (14/33, 42.4%) had an abnormal morphology. These abnormalities were characterized predominantly by multiple sperm penetration into the perivitelline space following conventional IVF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e) and cytoplasmic vacuoles at the pronuclear stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Five oocytes achieved fertilization; however, no viable embryos were obtained.\u003c/p\u003e\u003cp\u003eThe patient\u0026rsquo;s mother (Family2, I-2) with the same variant had given birth to four children at the ages of 22, 24, 30, and 32 years. She had no history of infertility treatment and experienced menopause at approximately 50 years of age. The patient\u0026rsquo;s sister (II-1) had conceived naturally at the age of 28 years without any infertility issues. Her other siblings (II-2 and II-3) were unmarried at the time of the analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVariant Analysis\u003c/h3\u003e\n\u003cp\u003eA genetic analysis identified heterozygous \u003cem\u003eLHX8\u003c/em\u003e variants in both probands (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In Family 1, the proband (II-1) carried the nonsense variant c.778C\u0026thinsp;\u0026gt;\u0026thinsp;T, which was not found in her mother (I-2). Since a paternal sample was unavailable, this variant indicated \u003cem\u003ede novo\u003c/em\u003e or paternal inheritance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In Family 2, the proband (II-4) carried the heterozygous splice site variant c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A, which was inherited from her mother (I-2). Sanger sequencing confirmed that the proband\u0026rsquo;s sister (II-1) and her son (III-1) were both negative for this variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverview of \u003cem\u003eLHX8\u003c/em\u003e variants\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eVariants\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eVariant type\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eZygosity\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003egnomAD\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eToMMo\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eACMG criteria\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eACMG pathogenicity class\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily 1 (Ⅱ-1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.778C\u0026thinsp;\u0026gt;\u0026thinsp;T(p.Q260*)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNonsense\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHeterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePVS1, PM2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLikely Pathogenic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily 2 (Ⅱ-4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ec.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSplicing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHeterozygous\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePVS1, PM2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLikely Pathogenic\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eNA, not available\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe nonsense variant c.778C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Gln260Ter) in Family 1 introduced a premature termination codon in exon 7. Regarding the splice site variant c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A in Family 2, a sequence analysis confirmed that it affected the invariant G nucleotide at the \u0026minus;\u0026thinsp;1 position of the canonical splice acceptor site. This nucleotide position showed complete conservation across vertebrate species in the \u003cem\u003eLHX8\u003c/em\u003e gene. Both variants were absent from population databases (gnomAD and ToMMo), indicating that they are not common polymorphisms in the general population.\u003c/p\u003e\u003cp\u003eTo examine the pathogenicity of the \u003cem\u003eLHX8\u003c/em\u003e variants in the present study, we utilized complementary metrics from multiple databases. \u003cem\u003eLHX8\u003c/em\u003e demonstrated an intolerance to loss-of-function variants, with gnomAD pLI of 0.97 and LOEUF of 0.52. A DECIPHER analysis indicated pHaplo of 0.75 and pLOF of 0.655.\u003c/p\u003e\u003cp\u003eRegarding the splice site variant c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A, SpliceAI predicted high probabilities of acceptor loss (0.99) and splice loss (0.85). Additionally, this variant showed a significant acceptor gain score (0.94) 2 bp from the original site, suggesting the activation of a cryptic splice site.\u003c/p\u003e\u003cp\u003eAccording to the ACMG/AMP guidelines, both variants were classified as likely pathogenic, fulfilling criteria PVS1 and PM2 in both cases (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eFunctional Analysis of the c.581-1G \u003e A Splice Site Variant\u003c/h3\u003e\n\u003cp\u003eTo investigate the functional consequences of the c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A variant on \u003cem\u003eLHX8\u003c/em\u003e splicing, we performed minigene splicing assays using the Exontrap system. The genomic fragment containing \u003cem\u003eLHX8\u003c/em\u003e exons 6 and 7 with the intervening intron 6 was cloned into the pET01 vector and transfected into HEK293 cells. The RT-PCR analysis of transfected cells revealed distinct splicing patterns between the wild-type (WT) and patient (PT) constructs. The WT minigene produced the expected transcript containing exons 6 and 7, with the normal splicing of intron 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, lane 2). No clear differences were detected in PCR product sizes between WT and PT constructs by agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Sanger sequencing of RT-PCR products revealed that the c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A variant resulted in the use of an aberrant splice site located 1 bp downstream of the original acceptor site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). This led to a 1-bp deletion in the mature transcript from the WT sequence. This splicing pattern was consistently observed across three independent experiments and aligned with SpliceAI predictions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe identified two women with primary infertility who carry novel \u003cem\u003eLHX8\u003c/em\u003e variants, each with high percentages of degenerated and immature oocytes. Despite different variants, the cases showed two common features: accumulation of sperm within the perivitelline space and increased cytoplasmic vacuoles in oocytes at the pronuclear stage.\u003c/p\u003e\u003cp\u003eThese morphological abnormalities may be understood in the context of the function of \u003cem\u003eLHX8\u003c/em\u003e. \u003cem\u003eLHX8\u003c/em\u003e acts as a key transcription factor in early oogenesis and interacts with \u003cem\u003eSOHLH1\u003c/em\u003e to form a nuclear complex [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This complex coordinates the expression of downstream genes that are essential for oocyte growth and differentiation, including \u003cem\u003eZp1\u003c/em\u003e and \u003cem\u003eZp3\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Zona pellucida proteins form the glycoprotein matrix around the oocyte and have central roles in species specific sperm recognition, binding, and prevention of polyspermy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The observed sperm accumulation suggests zona pellucida dysfunction, which may reflect altered expression or function of these proteins. Cytoplasmic vacuoles are established markers of reduced oocyte competence and are associated with lower fertilization rates and poorer outcomes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Collectively, these abnormalities support the view that \u003cem\u003eLHX8\u003c/em\u003e regulates oocyte quality through multiple pathways. Further functional studies are needed to clarify the diverse effects of \u003cem\u003eLHX8\u003c/em\u003e variants in oogenesis.\u003c/p\u003e\u003cp\u003eOur results extend previous reports on \u003cem\u003eLHX8\u003c/em\u003e related infertility. Zhao et al. described heterozygous loss of function \u003cem\u003eLHX8\u003c/em\u003e variants, including splicing, nonsense, and frameshift variants, caused human infertility [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The type of variant may modify the phenotype, as missense variants have been reported in patients with POI [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and common polymorphisms have been associated with the risk of POI in genome-wide studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In our study, both patients carried null variants and had normal ovarian reserve, with infertility as the only clinical finding.\u003c/p\u003e\u003cp\u003eThe minigene assay confirmed the use of a cryptic splice acceptor site 2 bp downstream, resulting in a 1 bp deletion and frameshift as predicted by SpliceAI. Despite this functional evidence, incomplete penetrance was evident in family 2, where the mother carried the same variant and was fertile. The pedigree, in which the proband's sister and her child did not inherit the variant and had normal fertility, supports incomplete penetrance. This has practical implications for counseling because clinical expression can be modified by trans-acting factors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], epigenetic changes, and environmental factors such as age and hormonal prolife.\u003c/p\u003e\u003cp\u003eThese observations also support interspecies differences in dosage sensitivity. While homozygous deletion of \u003cem\u003eLhx8\u003c/em\u003e results in a phenotype in mice, heterozygous \u003cem\u003eLHX8\u003c/em\u003e variants appear to be sufficient to cause reproductive abnormalities in humans [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This pattern, together with high constraint metrics such as a pLI of 0.97 and a pHaplo of 0.75 in our cases, suggests that human oogenesis is sensitive to \u003cem\u003eLHX8\u003c/em\u003e dosage. At the cellular level, recent work in mice links Lhx8 deficiency to altered autophagy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The vacuoles we observed may reflect a related disruption in cellular quality control.\u003c/p\u003e\u003cp\u003eClinically, our data suggest that \u003cem\u003eLHX8\u003c/em\u003e testing may be considered for women with recurrent OMA, particularly when cytoplasmic vacuoles or abnormal perivitelline sperm accumulation are present.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe report two clinical cases with novel LHX8 variants (c.778C\u0026thinsp;\u0026gt;\u0026thinsp;T and c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A) that are likely pathogenic according to ACMG and AMP guidelines. The consistent morphological and developmental findings support the central role of \u003cem\u003eLHX8\u003c/em\u003e in oocyte maturation. These features may guide genetic testing and counseling for unexplained infertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cp\u003eEthical approval was obtained from the Institutional Review Board of the Fujita Health University (HG24-014). Written informed consent was obtained from both participants prior to their inclusion in this study.\u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests in relation to this study.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP25ek0109760h0002.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors participated in the study\u0026rsquo;s design and revisions to the manuscript. Each author approved the final version and takes responsibility for all aspects of the work. Specifically, YS, HI, and HK conceptualized and designed the study. RY and HN provided critical feedback and helped in shaping the research, analysis, and manuscript. KK, YM, and TH contributed to the interpretation of clinical data. KN, KY, and RH performed laboratory experiments and data collection. HN and HK supervised the project, provided expertise for data interpretation, and secured funding. YS, HI, and HK drafted the initial manuscript and created the tables and figures.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiang Y, Huang J, Zhao Q, Mo H, Su Z, Feng S, et al. Global, regional, and national prevalence and trends of infertility among individuals of reproductive age (15\u0026ndash;49 years) from 1990 to 2021, with projections to 2040. Hum Reprod. 2025;40:529\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHatırnaz Ş, Hatırnaz ES, Ellibeş Kaya A, Hatırnaz K, Soyer \u0026Ccedil;alışkan C, Sezer \u0026Ouml;, et al. Oocyte maturation abnormalities - A systematic review of the evidence and mechanisms in a rare but difficult to manage fertility pheneomina. Turk J Obstet Gynecol. 2022;19:60\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Der Kelen A, Uyttebroeck S, Van de Voorde S, Picchetta L, Segers I, Vlaeminck J, et al. Oocyte/zygote/embryo maturation arrest: a clinical study expanding the phenotype of NOBOX variants. J Assist Reprod Genet. 2025;42:763\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng R, Sang Q, Kuang Y, Sun X, Yan Z, Zhang S, et al. Mutations in TUBB8 and Human Oocyte Meiotic Arrest. N Engl J Med. 2016;374:223\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKitanaka J, Takemura M, Matsumoto K, Mori T, Wanaka A. Structure and chromosomal localization of a murine LIM/homeobox gene, Lhx8. Genomics. 1998;49:307\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFu L, Zhang M, Mastrantoni K, Perfetto M, Wei S, Yao J. Bovine Lhx8, a germ cell-specific nuclear factor, interacts with Figla. PLoS ONE. 2016;11:e0164671.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi Y, Ballow DJ, Xin Y, Rajkovic A. Lim homeobox gene, lhx8, is essential for mouse oocyte differentiation and survival. Biol Reprod. 2008;79:442\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao L, Li Q, Kuang Y, Xu P, Sun X, Meng Q, et al. Heterozygous loss-of-function variants in LHX8 cause female infertility characterized by oocyte maturation arrest. Genet Med. 2022;24:2274\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBolor H, Mori T, Nishiyama S, Ito Y, Hosoba E, Inagaki H, et al. Mutations of the SYCP3 gene in women with recurrent pregnancy loss. Am J Hum Genet. 2009;84:14\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Z, Liu C-Y, Zhao Y, Dean J, FIGLA. LHX8 and SOHLH1 transcription factor networks regulate mouse oocyte growth and differentiation. Nucleic Acids Res. 2020;48:3525\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePangas SA, Choi Y, Ballow DJ, Zhao Y, Westphal H, Matzuk MM, et al. Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8. Proc Natl Acad Sci U S A. 2006;103:8090\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWassarman PM. Zona pellucida glycoproteins. J Biol Chem. 2008;283:24285\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNikiforov D, Gr\u0026oslash;ndahl ML, Hreinsson J, Andersen CY. Human oocyte morphology and outcomes of infertility treatment: A systematic review. Reprod Sci. 2022;29:2768\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBouilly J, Beau I, Barraud S, Bernard V, Azibi K, Fagart J, et al. Identification of multiple gene mutations accounts for a new genetic architecture of primary ovarian insufficiency. J Clin Endocrinol Metab. 2016;101:4541\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin Y, Zhao H, Kovanci E, Simpson JL, Chen Z-J, Rajkovic A. Analysis of LHX8 mutation in premature ovarian failure. Fertil Steril. 2008;89:1012\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCooper DN, Krawczak M, Polychronakos C, Tyler-Smith C, Kehrer-Sawatzki H. Where genotype is not predictive of phenotype: towards an understanding of the molecular basis of reduced penetrance in human inherited disease. Hum Genet. 2013;132:1077\u0026ndash;130.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastel SE, Cervera A, Mohammadi P, Aguet F, Reverter F, Wolman A, et al. Modified penetrance of coding variants by cis-regulatory variation contributes to disease risk. Nat Genet. 2018;50:1327\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRen Y, Suzuki H, Jagarlamudi K, Golnoski K, McGuire M, Lopes R, et al. Lhx8 regulates primordial follicle activation and postnatal folliculogenesis. BMC Biol. 2015;13:39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026rsquo;Ignazio L, Michel M, Beyer M, Thompson K, Forabosco A, Schlessinger D, et al. Lhx8 ablation leads to massive autophagy of mouse oocytes associated with DNA damage. Biol Reprod. 2018;98:532\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LHX8, oocyte degeneration, maturation arrest, primary infertility, Oocyte/Zygote/Embryo Maturation Arrest (OZEMA)","lastPublishedDoi":"10.21203/rs.3.rs-7460153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7460153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLHX8\u003c/em\u003e gene encodes a germ cell specific transcription factor that is required for oocyte development. We evaluated two unrelated women with primary infertility who showed reproducible oocyte abnormalities across in vitro fertilization cycles, and we performed genomic and functional assays to clarify the role of \u003cem\u003eLHX8\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWhole exome sequencing identified heterozygous loss-of-function variants in \u003cem\u003eLHX8\u003c/em\u003e (NM_001001933.1) in both patients: c.778C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Gln260Ter) in family 1 and c.581-1G\u0026thinsp;\u0026gt;\u0026thinsp;A in family 2. Both variants met the American College of Medical Genetics and Genomics criteria for likely pathogenicity. The two patients had high proportions of degenerated or immature oocytes and showed consistent morphologic features, including multiple cytoplasmic vacuoles, impaired zona pellucida function with accumulation of sperm in the perivitelline space, and poor embryo development. The splice site variant was inherited from a fertile mother, which indicates incomplete penetrance. A minigene assay confirmed the use of a cryptic acceptor site that produced a one nucleotide deletion and a frameshift, consistent with loss of function.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThese findings expand the phenotypic spectrum of \u003cem\u003eLHX8\u003c/em\u003e related infertility and provide mechanistic evidence that partial reduction of \u003cem\u003eLHX8\u003c/em\u003e activity compromises oocyte quality. Recognition of the characteristic morphology may guide genetic testing and counseling in cases of unexplained infertility.\u003c/p\u003e","manuscriptTitle":"Novel LHX8 variants associated with distinctive oocyte morphological abnormalities and maturation arrest in primary infertility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 08:43:06","doi":"10.21203/rs.3.rs-7460153/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-06T18:16:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T22:08:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-03T19:27:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13714230704966120468583463222177474177","date":"2025-10-08T14:41:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116917472967192104945071170309479020523","date":"2025-10-07T22:19:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-07T21:58:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-09T01:19:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-08T04:49:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2025-08-26T07:40:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7bed4490-51e9-48f4-8615-84d49e669fc1","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:03:01+00:00","versionOfRecord":{"articleIdentity":"rs-7460153","link":"https://doi.org/10.1186/s13048-026-01978-2","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2026-01-21 15:58:21","publishedOnDateReadable":"January 21st, 2026"},"versionCreatedAt":"2025-10-20 08:43:06","video":"","vorDoi":"10.1186/s13048-026-01978-2","vorDoiUrl":"https://doi.org/10.1186/s13048-026-01978-2","workflowStages":[]},"version":"v1","identity":"rs-7460153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7460153","identity":"rs-7460153","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}