Genetic and physiological study of morphologically abnormal human zona pellucida.

OBJECTIVE: To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance. STUDY DESIGN: The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed. RESULTS: No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3. CONCLUSIONS: The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.

[The involvement of the zona pellucida in unexplained infertile women].

Unexplained infertility (*UI) was a common problem before the IVF era. A couple was declared as UI only after they passed all the routine common tests and no reason was found for the infertility. The introduction of ICSI in the IVF clinics enabled the investigation of the quality of the couple's gametes. Thus, the definition of the term UI was refined and the number of couples encompassed by this term decreased dramatically. Zona Pellucida (ZP) incompetence was described as one of the causes of UI. We recently (2004) published a case study of an UI couple due to this cause. The oocytes of the woman collapsed during the preparation of the oocytes for sperm injection. These oocytes suffered from irregular ZP and abnormal appearance. Only after gentle treatment of the oocyte-cumulus complex and ICSI fertilization, embryo development and delivery of normal newborn was achieved. This woman and others who suffer from UI did not conceive in a natural way since the ZP of the ovulated eggs did not bind sperm cells and, thereby, were not fertilized. It only recently became possible to find the reason for the infertility of the couple and the solution, through the use of highly advanced technology in the IVF-ICSI process. One of the reasons for the deformatted ZP is the malfunction of the gene that encodes the 4 glycoproteins, which compose the ZP. This is now under investigation in our Institute.