Causes And Therapeutic Strategy Of Fertilization Failure In Globozoospermic Patients And Establishment Of Genetic Diagnosis Platform For The Disease

Globozoospermia is a rare （incidence<0.1%） but severe form of teratozoospermia causing primary male infertility, characterized by the production of a majority of the round-headed spermatozoa lacking an acrosome. Due to the absence of acrosome, round-headed spermatozoa fail to penetrate the zona pellucida resulting in fertilization failure, thus causing male infertily.Traditionally, the round-headed spermatozoa could be treated with the help of Intracytoplasmic sperm injection （ICSI）. However, low fertilization or fertilization failure after ICSI was frequently observed. Recent studies suggested that fertilization failure in globozoospermic patients could be associated with the decrease or defect of phospholipase C （PLCζ）, a protein involved in the induction of calcium oscillations triggering oocyte activation. PLCζ was variably detectable in three localities within the perinucler theca （PT） of sperm head:the equatorial segment and acrosomal/post-acrosomal region. The mutations of PLCζ were found in one non-globozoospermic patient also suggested the role of PLCζ in the induction of calcium oscillations triggering oocyte activation.Recent studies reported that fertilization and pregnancy rates could be improved after ICSI with assisted oocyte activation （AOA）, and no abnomalies were found in these children.Globozoospermia was divided by the ejaculates that consists of100%round-headed spermotozoa lacking an acromosome （Type â…  globozoospermia）, or contains<100%of globozoospermic cells （Type â…¡ globozoospermia）. At present, there are different options of treatment according the types of Globozoospermia:ICSI and AOA were mainly used in Type I globozoospermic patients, while for Type â…¡ globozoospermic patients, normal morphology spermatozoa could be chosed in the ejaculates for ICSI. Recent studies found a very low percentage of spermatozoa with abnormal acrosomes （atrophied, misplaced and a small bud of acrosome） in the ejaculates of globozoospermic patients by using transmission electron microscopy （TEM） and the analysis of immunoflurescence. Successful childbirth after ICSI by using the spermatozoa with the small bud of acrosome without assisted oocyte activation in a patient with globozoospermia. Although assisted oocyte activation could improve the fertilization rates after ICSI, the safety of the optition was still worried. It would be valuable to test fertilization, pregnancy, childbirth and birthdefect of the above "round-headed spermatozoa" with abnormal acrosomes by retrospective or prospective study in order to offer more efficient and safety trement option for type I globozoospermic patients.There are more case reports about globozoospermia since the first globozoospermia report by Schirren C et al in1971, which were mainly focused on morphological description and etiology of globozoospermia. The pathogenesis of globozoospermia is unclear, but the report of globozoospermic brothers suggested the globozooseprmia is one kind of genetic diseases, which was more accepted after the reports of several kinds of model mouse showing similar phenotype to globozoospermia.The characteristic phenotype of globozoospermia being only an absence of acrosome implied a single gene disese more probably, which made the indetifation of candidate genes for globozoospermia attractive. The progress on cloning the gene for globozoospermia was very slowly due to the rare and sporadic cases. With the emergence of new genetic technologies, such as microarry analysis and genome sequencing, two candidate genes for human globozoospermia were indentified and the previous studies on model mouse made the gentic diagnosis possible resulting in facilitating an adequate genetic counseling, which prevented the offspring from globozoospermia. The purpose of the study is to discuss the possible causes and therapeutic strategy of fertilization failure in globozoospermic patients and to establish the genetic diagnosis platform for the disease.Part â…  Causes and Therapeutic Strategy of Fertilization Failure in Globozoospermic PatientsFirstly, we investigate the clinical data of globozoospermic patients recruited in our hospital between January2007and September2012. Fertilization, pregnancy and childbirth rates of conventional ICSI, ICSI with AOA and aritificial insemination by donor or in vitro fertilization by donor were statistically analyzed. One male birth after ICSI without AOA in one globozoospermic patient and one female birth after ICSI with AOA in one globozoospermic patient were observed. Peripheral blood and semen samples from two additional cases of mini-acrosome spermatozoa （including one miniacrosome spermatozoa patient with recurrent IVF failure） and one additional case of normal morphology spermatozoa with recurrent ICSI failure were also recruited. Secondly, we carefully analyzed semen samples of the above two globozoospermic patients with childbirth, one patient with miniacrosome and one patient with normal morphology spermatozoa but with recurrent ICSI fertilization failure by using papanicolaou staining, electronic microscopy and immunofluorescence. We then discussed the causes of fertilization failure in order to offer efficient and safety treatment option for those patients.Part â…¡ Establishment of Genetic Diagnosis Platform of GlobozoospermiaAlthough there were many candidate genes for globozoospermia reported, there are few mutations identified in these genes, most globozoospermic patients got no clear genetic diagnosis. Some previous studies suggested that DPY19L2is the major cause of globozoospermia. The molecular defect of DPY19L2is recurrently homozygous deletion of the whole DPY19L2gene resulting from non-allelic homologous recombination （NAHR）. In addition, DPY19L2point mutations are also the cause of globozoospermia. There exist several pseudogenes of DPY19L2so that the gentic analysis of DPY19L2becomes very difficult, and thus it requires special care for the specificity of primers, the stability of PCR conditions and analysis of sequencing results. In the study, we recruited a total of17globozoospermic patients. Molecular analysis for deletions and mutations in the DPY19L2gene was performed on the corresponding16genetically independent individuals. Four of the16genetically independent patients with globozoospermia were homozygous for the DPY19L2deletion, six were homozygous for a point mutation including a nucleotide deletion c.1532delA in two individuals, a multi-mutation consisting of a nucleotide deletion c.1679delT and a two-nucleotide deletion c.1681₁682delAC （c.[1679de1T;1681₁682delAC]） in one patient, a recurrent missense mutation c.869G>A in one individual and a missense mutation c.989T>C in one patient, one missense mutation c.1533G>T, and one additional patient had a heterozygous deletion in one allele but with no mutation identified in another allele, the remain five patients were identified without deletion or mutation. Overall,62.5%of the patients （n=16） have a sequence variant of DPY19L2in both alleles. This study confirms again that the DPY19L2mutations are the major cause of globozoospermia. Four novel mutations were found in this study, further broadening the spectrum of DPY19L2mutations. By setting up the genetic diagnostic platform for DPY19L2in globozoospermic patients, most patiets got a clear genetic diagnosis and it would facilitate genetic counseling.