Introduction
Human reproduction is a complex and tightly regulated biological process involving gamete maturation, fertilization, embryo development, blastocyst formation, implantation, and ultimately, live birth. Disruption or arrest at any of these stages can result in reproductive failure.1
Infertility, defined as the inability to conceive after one year of regular, unprotected intercourse, affects an estimated 48 million couples worldwide.2,3 Debate exists around the classification of infertility into primary and secondary types, but both contribute significantly to global reproductive health challenges. As infertility rates rise, increasing numbers of couples are turning to assisted reproductive technologies (ART), such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), success rates remain suboptimal, partly due to underlying genetic factors. Genetic abnormalities have been implicated in up to that contribute up to 30% of infertility cases.4,5
Female fertility is critically dependent on successful oogenesis where immature oocytes undergo two meiotic divisions (meiosis I and II) to produce haploid gametes.6 The oocyte arrest at meiosis II and completed the process when fertilization take place.7,8
Oocyte maturation involves two essential components: nuclear maturation, referring to chromosomal reduction via meiosis, and cytoplasmic maturation, which prepares the oocyte for fertilization, activation, and early embryonic development.9
The WEE2 gene (WEE1 homolog 2), located on chromosome 7q34, encodes a 567-amino acid oocyte-specific kinase belonging to the WEE family of protein kinases. It consists of 12 exons and is exclusively expressed in oocytes and zygotes.10 WEE2 plays a vital role in maintaining meiotic arrest at the GV stage by inhibiting maturation-promoting factor (MPF) through CDK inactivation. Additionally, WEE2 is required for exit from metaphase II during fertilization.11 Reduced expression of WEE2 has been associated with elevated MPF activity and impaired MII exit, leading to failure in pronucleus formation and fertilization.12
Given the critical role of WEE2 in oocyte maturation and fertilization, this study aimed to assess the association between WEE2 gene polymorphism and fertilization failure in women undergoing ICSI. To our knowledge, this represents the first study in Iraq investigating the relationship between WEE2 genetic variation and fertilization outcomes.
Materials and Methods
Study Design and Setting
This prospective comparative study was conducted between October 2024 and February 2025 at Tibacenter for Infertility in Babil, Iraq. A total of 137 infertile women undergoing intracytoplasmic sperm injection (ICSI) were enrolled.
Study Population
Eligible participants were women aged 20–35 years with primary or secondary infertility of 2–10 years’ duration. All participants were undergoing ICSI cycles. Inclusion criteria encompassed women with no history of systemic diseases or chromosomal abnormalities. All male partners underwent standard semen analysis.
Clinical and Laboratory Evaluation
Comprehensive gynecological and general examinations were performed. Baseline fertility assessments included hormonal profiling on day 1 or 2 of the menstrual cycle, measuring FSH, LH, estradiol (E2), and progesterone (P4). Transvaginal ultrasound was performed on cycle day 2 to assess baseline ovarian status.
Controlled Ovarian Stimulation and ICSI Procedure
All patients underwent ovarian stimulation using a flexible GnRH antagonist protocol. Oocyte retrieval was performed 34–36 hours after administration of the ovulation trigger via transvaginal ultrasound-guided aspiration. Semen samples were collected on the same day and processed for ICSI.
The ICSI procedure involved microinjection of a single spermatozoon into the cytoplasm of each mature oocyte. Fertilization was confirmed 16–18 hours post-injection by the presence of two pronuclei (2PN) and two polar bodies (2PB). Abnormal fertilization was defined by the presence of 1PN, 3PN, or 4PN.
Embryo Evaluation and Transfer
Embryo grading was performed on day 3 based on standard morphological criteria:
Grade I: <10% fragmentation, stage-specific cell size, no multinucleation.
Grade II: 10–25% fragmentation, mostly stage-specific cell size, no multinucleation.
Grade III: >25% fragmentation, irregular cell size, presence of multinucleation.
Embryos graded I and II were selected for uterine transfer based on maternal age, embryo quality, and IVF history.
Molecular Analysis
Sample Collection and DNA Extraction
Peripheral venous blood (2 mL) was collected into EDTA tubes and stored at −20°C until analysis. Genomic DNA was extracted using a standard phenol–chloroform protocol. DNA quantity and purity were assessed via NanoDrop spectrophotometry (BioDrop, UK), with absorbance ratios at 260/280 and 260/230. DNA integrity was confirmed by agarose gel electrophoresis.
Primer Design
Primers for rs1476640 of the WEE2 gene were designed using NCBI Primer-BLAST and validated for specificity using BLAST against the human genome. Secondary structure and self-complementarity were assessed using OligoCalc (Kibbe, 2007). Amplicon verification was performed using MFEprimer-2.0 (Qu et al, 2012). Primer sequences and characteristics are provided in Table 1.
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Table 1 Primer Sequences and Properties Used for Amplifying the WEE2 Gene (rs1476640 SNP)
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PCR and Genotyping
PCR Amplification
PCR was optimized for annealing temperature and cycle number. Each 20 μL reaction included 8 μL 2X Master Mix, 1 μL forward primer (10 pmol), 1 μL reverse primer (10 pmol), 2 μL genomic DNA (10–20 ng/μL), 0.5 μL MgCl2 (1%), and 7.5 μL nuclease-free water. The amplified product was confirmed on agarose gel prior to restriction digestion.
RFLP Analysis
The rs1476640 SNP was genotyped using NdeI enzyme digestion. The 20 μL reaction mix included 5 μL PCR product, 1.5 μL enzyme buffer, 0.5 μL NdeI, and 8 μL ddH2O. Fragments were separated on 2.5% agarose gel and visualized under UV light.
Statistical Analysis
Statistical analyses were performed using SPSS version 26, Microsoft Excel 2010, and SNPStats (Sole et al, 2006). Independent samples t-tests were used for normally distributed continuous variables. The Kruskal–Wallis test was used for non-parametric comparisons. Chi-square tests assessed associations between categorical variables. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the association between genotype and fertilization outcomes.
Ethical Approval
This study was approved by the Ethics Committee of the University of Babylon, Hammurabi College of Medicine, Iraq (Document No. 40, dated September 15, 2024). All participants provided informed verbal and written consent in accordance with the Declaration of Helsinki.
Results
Demographic Characteristics of Women Enrolled in the Present Study
The demographic characteristics of infertile patients are shown in Table 2. The Mean, Standard Deviation and Standard Error of female age with positive and negative pregnancy were 30.73, 5.025 and 0.649, and 29.88, 5.385 and 0.614, respectively, and the age was ranging from 20 to 39years and 18 to 39 years, respectively, and there was no significant difference (p = 0.347).
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Table 2 Demographic Characteristics of Female Enrolled in the Present Study
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In addition, there was no significant difference in mean of BMI between positive pregnancy group and negative pregnancy groups (p = 0.271).
Regarding the duration of infertility, there was significant difference between positive pregnancy and negative pregnancy groups (p = 0.038). The Mean, Standard Deviation and Standard Error of Duration with positive and negative pregnancy were 6.90, 4.387 and 0.566, and 5.43, 3.833 and 0.437, respectively.
Comparison of Total Oocyte Count Between Positive Pregnancy Group and Negative Pregnancy Group
The Mean and Standard Deviation of total oocyte count with positive and negative pregnancy were 12.67 and 4.990 and 10.82 and 5.241, respectively, and the Standard Error was 0.644 and 0.597), respectively; the result showed significant association between pregnancy group and negative pregnancy group (0.038) Table 3.
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Table 3 Comparison of Total Oocyte Count Between Positive Pregnancy Group and Negative Pregnancy Group
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Comparison of Endometrial Thickness Between Positive Pregnancy Group and Negative Pregnancy Group
The Mean and Standard Deviation of Endometrial thickness with positive and negative pregnancy were 11.84 and 2.763, and 10.83 and 2.897, respectively, and the Standard Error was 0.357, and 0.330) respectively; the difference in mean rank was statistically significant (p = 0.04) Table 4.
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Table 4 Comparison of Endometrial Thickness Between Positive Pregnancy Group and Negative Pregnancy Group
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Amplification of WEE2 Gene
In this study, the PCR technique was used to detect WEE2 gene. Gel electrophoresis results indicated the size of the amplicons as 329 bp for WEE2 gene, compared with the DNA ladder (Figures 1–2). The genotypes of SNP rs1676760 were determine by PCR-RFLP method (Figure 3).
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Figure 1 Optimization of PCR by gradient annealing temperature. Lane L 100bp step DNA ladder; other lanes represent different annealing temperature as indicated on each lane.
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Figure 2 PCR amplification of 329bp amplicon for rs1476640 genotyping. The product was resolved on 2% agarose gel. lane L 100bp step DNA ladder. This product was subjected to restriction enzyme analysis.
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Figure 3 Genotyping of rs1476640 polymorphism by PCR-RFLP technique. PCR product were digested by Ndel restriction enzyme lanes L 100bp step DNA ladder: lanes 1, 5 and 13 show the TT genotype; lanes 2, 9, 12 and 14 show the TC genotype; lanes 3, 4, 6, 7, 8, 10 and 11 show the CC genotype.
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Genetic Association of T and C Alleles Between the Positive Pregnancy Group and Negative Pregnancy Group
The results showed that there was significant allele frequency difference between the positive pregnancy group and negative pregnancy group (Table 5).
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Table 5 Genetic Association of T and C Alleles Between the Positive Pregnancy Group and Negative Pregnancy Group
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Association of rs1476640 Genotypes with Pregnancy Outcome in Female Under ICSI
The difference in TT between positive and negative pregnancy was statistically highly significant Codominance TT (reference) is 50.6% and 18.3%, respectively, C/T is 37.7%, and 43.3%), respectively, and C/C is 11.7%, 38.3%, respectively (p = < 0.0001). Dominant C/ T-C/C 39 (50.6%), 49 (81.7%), respectively (p = 1e-04). Recessive C/C 9 (11.7%), 23 (38.3%), respectively (p = 2e-04) (Table 6).
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Table 6 Association of rs1476640 Genotypes with Pregnancy Outcome in Female Under ICSI
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Comparison of Oocyte Maturation and Genotype Between the Positive Pregnancy Group and Negative Pregnancy Group
There was significant difference in Germinal vesicle oocyte between positive and negative pregnancy median (IQR) CC 3 (2), CT 0 (2), TT 0 (2) respectively; (p < 0.001). In addition there was no significant difference in (metaphase I, MI) oocytes between pregnant and non pregnant ladies (Table 7).
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Table 7 Comparison of Oocyte Maturation and Genotype Between the Positive Pregnancy Group and Negative Pregnancy Group
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In addition, there was significant difference in the number of germinal vesicle (GV) oocytes in pregnant ladies (p < 0.001). There was also no significant difference in the number of immature (metaphase I, MI) oocytes in pregnant.
A statistically significant difference in Germinal vesicle oocyte in nonpregnant ladies (p < 0.001). However, the stage of oocyte maturation includes metaphase I (MI) which showed no significant difference in negative group.
Discussion
This study investigated the association between WEE2 gene polymorphism and fertilization outcomes in infertile women undergoing intracytoplasmic sperm injection (ICSI). The findings revealed no statistically significant difference in the mean age or BMI between pregnant and non-pregnant groups, consistent with previous studies that suggest age and BMI may not be sole predictors of ICSI success in populations where cultural norms encourage early marriage.13–15
However, the duration of infertility showed a significant association with pregnancy outcome, in agreement with prior studies indicating that prolonged infertility may negatively impact clinical success rates in ART procedures.16
Importantly, the number of mature oocytes retrieved was significantly higher in the pregnant group, reinforcing the well-established link between oocyte yield and fertilization success.17 Endometrial thickness also showed a significant association with pregnancy, with thicker endometria correlating with improved implantation rates, consistent with findings from earlier studies.18,19
Beyond clinical parameters, our study highlights the critical role of genetic factors, specifically WEE2 gene polymorphisms, in determining ICSI outcomes. The codominant model revealed a higher frequency of C/C and C/T genotypes in non-pregnant women, while the T/T genotype was more common in those who achieved pregnancy, suggesting a protective role of the T allele and a pathogenic effect of the C allele. These observations are in line with previous reports that have linked WEE2 mutations to total fertilization failure (TFF) due to their impact on oocyte maturation and MII exit.20–22
WEE2 encodes an oocyte-specific kinase that regulates meiotic arrest at the GV stage and is essential for exit from metaphase II following fertilization. Mutations or reduced expression of WEE2 impair this process, resulting in abnormal oocyte activation and fertilization failure.23,24 Studies have shown that up to 20.8% of women with TFF carry WEE2 mutations, while other investigations report a prevalence of approximately 6%.24–26 Our findings support this association and emphasize the gene’s role in regulating oocyte competence.
Furthermore, the data are consistent with reports by Jin et al (2021), who identified compound heterozygous WEE2 mutations in patients with unexplained infertility, and other studies confirming that WEE2 defects can halt oocyte development at the GV stage or block MII progression.24,27
Collectively, these findings underscore the importance of WEE2 in human oocyte development, meiotic regulation, and successful fertilization. Identifying polymorphisms in this gene could enhance early diagnosis of genetic infertility, inform patient counseling, and potentially guide personalized treatment strategies in ART.
Conclusion
Polymorphisms in the WEE2 gene, particularly the rs1476640 variant, are significantly associated with fertilization failure in women undergoing ICSI. The presence of the C allele may impair oocyte maturation by disrupting the meiotic process, thereby reducing the fertilization potential. Genetic screening for WEE2 variants may offer a valuable tool for identifying patients at risk of ART failure and improving individualized infertility treatment approaches.
Disclosure
The authors report no conflicts of interest in this work.
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