Researchers determine 14 variants in the egg’s protective layer associated with female infertility which can affect assisted reproductive technology outcomes

While assisted reproductive technologies have improved outcomes for many patients, some women still experience persistent fertilization failure. This may be attributed to gene variants of zona pellucida (ZP) proteins which play critical role in sperm binding and embryo development. To elucidate this, researchers from China conducted genetic analysis in women with primary infertility. They identified rare ZP gene variants linked to fertilization failure and implantation challenges, highlighting the importance of early screening and personalized treatment.

Infertility affects millions of women worldwide. While assisted reproductive technologies (ART) such as in vitro fertilization (IVF) is helping patients conceive, some women continue to experience unexplained treatment failure. During IVF treatments, eggs are stimulated to mature within ovarian follicles and are then retrieved for fertilization in the laboratory. However, in some cases, the follicles appear to be normal but no eggs are retrieved, a condition known as empty follicle syndrome.

A key structure involved in fertilization is the zona pellucida (ZP), the egg’s protective outer layer. The proteins of this layer play important roles in sperm binding and embryo development. It is anticipated that genetic defects affecting ZP may contribute to certain cases of infertility.

To elucidate this, a team of researchers led by Professor Qian-hong Ma from the Department of Obstetrics and Gynecology and Key Laboratory of Obstetric, Gynecologic, and Pediatric Diseases and Birth Defects of Ministry of Education, Sichuan University, China, conducted a retrospective genetic analysis in women with infertility. This study was made available online on February 11, 2025, and was published in Volume 132, Issue S2 of BJOG: An International Journal of Obstetrics & Gynaecology on April 15, 2025.

“To identify ZP variants associated with female infertility, we evaluated the clinical characteristics and pregnancy outcomes of patients with primary infertility, characterized by ZP abnormalities who underwent ART treatment,” says Prof. Ma.

The researchers analyzed DNA from blood samples of 12 women with primary infertility and persistent poor ART outcomes using advanced methods such as next-generation sequencing and Sanger sequencing.

“We identified 14 different genetic variants in three ZP genes, including three in ZP1, five in ZP2 and four in ZP3. 8 of these variants were novel and had not been reported previously,” reports Prof. Ma.

These genes produce structural proteins that form the ZP. The ZP3 protein helps sperms to recognize and bind to the egg, while ZP2 supports continued sperm interaction and ZP1 stabilizes the overall structure. Variants affecting these genes may therefore disrupt different stages of fertilization and early embryo development. The clinical observations confirmed these effects.

In several patients, immature or degenerated eggs were observed and were associated with ZP1 variants. In some cases, genuine empty follicle syndrome, where no eggs are retrieved despite apparently normal follicle growth on ultrasound, was also reported. Moreover, certain ZP1 variants were predicted to disrupt the formation of a functional ZP.

ZP2 variants were linked to structural abnormalities of the egg’s outer layer, including eggs lacking a ZP, enlarged spaces surrounding the egg, and abnormal sperm binding patterns. Such defects may interfere with fertilization by disrupting normal sperm–egg interaction.

Meanwhile, alterations in the ZP3 gene were associated with abnormal ZP appearance and poor egg retrieval, including eggs with a rigid or “waxy” outer layer. In some patients, multiple follicles developed but very few viable eggs were obtained, suggesting an important role for ZP3 in maintaining egg integrity during development.

Based on these findings, the researchers suggested certain strategies to overcome some of these challenges. For cases with zona pellucida abnormalities, they suggest intracytoplasmic sperm injection, a technique in which a single sperm is directly injected into the egg, so it may bypass the fertilization barriers. However, in cases where severe genetic defects affect the egg development, alternative approaches such as use of donor eggs may be needed.

Overall, the study reveals association between ZP gene variants and female infertility, highlighting the importance of early genetic testing in women experiencing repeated failures during ART. While the study offers important insights, its small sample size and retrospective design may limit how widely the findings apply. Further studies are needed to confirm these genetic effects and evaluate the role of genetic screening in fertility care.

“Through genetic screening, we can better identify underlying biological causes of infertility and guide more personalized treatment strategies and enhance fertility outcomes,” concludes Prof. Ma.

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Reference
Sa, S.-w., Wang, L.-l., and Ma, Q.-h. Association of Zona Pellucida Gene Variants with Female Infertility: A Retrospective Genetic Analysis. BJOG: An International Journal of Obstetrics & Gynaecology 132, 75–82 (2025).
https://doi.org/10.1111/1471-0528.18094


About Sichuan University, China
Sichuan University is located in Chengdu, China, founded in 1896. Sichuan University offers a complete range of 13 disciplines, covering literature, science, engineering, medicine, economics, management, law, etc., and boasts multiple state key laboratories, research bases, abundant teaching resources and a strong faculty team. The university ranks top in China in such research areas as natural sciences, engineering technology, humanities and social sciences, and medicine. In 2025, Sichuan University was ranked 87^th in Shanghai Ranking's Academic Ranking of World Universities.
Website: https://en.scu.edu.cn/


About Professor Qian-hong Ma from Sichuan University, China
Prof. Qian-hong Ma is affiliated to the Department of Obstetrics and Gynecology and Key Laboratory of Obstetric, Gynecologic, and Pediatric Diseases and Birth Defects of Ministry of Education, Sichuan University, China. Her research focuses on female infertility, assisted reproductive technology outcomes, reproductive genetics, and gynecology.

Funding information
The authors received no specific funding for this work.