Globozoospermic Sperm Research Articles

P-001 Comparing two monomorphic teratozoospermia syndromes in terms of ICSI outcomes - Round vs. pin-head

Abstract Study question Whether ART clinical outcomes are affected at extremes of morphological presentations, in samples where typical forms are unavailable for ICSI. Summary answer The study found no correlation between the severity of sperm defect and ICSI results. What is known already One of the challenging topics in the field of Andrology is the predictive role of monomorphic teratozoospermia, specifically head defects, in assisted reproductive technology (ART) outcomes. The definitive requirement of conducting intracytoplasmic sperm injection (ICSI) is the presence of at least one motile spermatozoon. Two schools of thought provide contradicting views; some believe that sperm morphology can significantly predict fertilization, clinical pregnancy, and live birth rates in fresh ICSI cycles and others support that the degree or nature of sperm morphological dysfunction lacks a significant impact on the outcome of ICSI outcomes. Study design, size, duration This retrospective cohort study was included 7,881 couples with male factor infertility who underwent 11,536 IVF/ICSI cycles at a tertiary referral fertility center over a 13-year period. Participants/materials, setting, methods From this cohort, 10 cases were identified with >90% acephalic sperm syndrome and 11 patients had >90% globozoospermic sperm. The implantation rate, pregnancy, and live birth rates per initiated cycle (iC) and per patient were compared between the two groups. The rates were also compared between each group with the general ICSI population. Main results and the role of chance The data analysis suggests no statistically significant differences between the two patient groups. Implantation was 20% versus 15.1% (95% CI, 0.33 to 5.97; OR, 1.4) for round and pin-head groups. The pregnancy per iC and patient were 25% vs 23.1% (95% CI, 0.25 to 9.5; OR, 1.55) and 27.3% vs 30% (95% CI, 0.13 to 5.8; OR, 0.87) for round-head and pin-head groups, respectively. The live birth rates matched the pregnancy rates. For the general ICSI population the rates were as follows: implantation rate was 17.3%; pregnancy rate (per iC and per patient) was 27.2% and 44.7%; and live birth rate (per iC and per patient) was 23.2% and 38.2%. The study found no significant differences between the outcomes for the general ICSI population and the two groups. Limitations, reasons for caution It is uncertain whether this lack of association is genuine or whether it is affected by the limited number of cases with extreme monomorphic abnormalities. The absence of evidence should not be taken as evidence of absence. Wider implications of the findings Conducting such studies should be encouraged to provide insight by informing systematic reviews. A significant portion of existing literature on teratozoospermia comprises of case reports, which are rationally excluded from systematic reviews and meta-analyses. Additional research is warranted to definitively establish the investigated relationships. Trial registration number not applicable

Proteomic Research of Molecular Defects in Globozoospermia.

Globozoospermia is a form of teratozoospermia characterized by round-headed spermatozoa and lack of acrosome. These spermatozoa cannot penetrate the zona pellucida of the oocyte, resulting in unsuccessful fertilization and infertility. When intracytoplasmic sperm injection is performed, the fertilization rate tends to be low. Until now, the causes of this disorder remain to be elucidated; however, mutations of some genes segregating on an autosomal recessive mode have been associated with this infertile condition. DPY19L2 (dpy-19-like 2 [Caenorhabditis elegans]) codes for a transmembrane protein expressed predominantly in spermatids, with specific localization limited to the internal nuclear membrane. Genetic defects in the DPY19L2 gene have been demonstrated the most frequent genetic cause of globozoospermia; however, intracellular molecular pathways related to its encoded protein are largely unknown. In this issue of Proteomics Clinical Applications, Guo and co-workers investigate the proteome of gloobozoospermic spermatozoa. The authors identified 491 proteins that are differentially expressed in globozoospermia (370 are upregulated and 121 are downregulated in DPY19L2-deficient globozoospermic sperm). Notably, the molecular defects identified by the authors are closely related to biological processes involved in acrosome formation, chromatin composition, sperm-egg binding, and fertilization.

Proteomic Analysis of Dpy19l2-Deficient Human Globozoospermia Reveals Multiple Molecular Defects.

To investigate the differences in protein expression between Dpy19l2-deficient human globozoospermia and normozoospermia. Human sperm samples from three globozoospermic donors with Dpy19l2 deletion and three normal controls are subjected to TMT quantitative technology. SPESP1, HIST1H4A, and LYZL1 are randomly selected for western blotting analysis. GO annotations are performed using the Database for Annotation, Visualization, and Integrated Discovery. A total of 2567 proteins are identified, of which 2510 proteins are quantified, and 491 are differentially expressed (fold-change >2), with 370 upregulated and 121 downregulated in globozoospermic patients. The levels of several important proteins, including SPACA 1, IZUMO1, ZPBP1, and PLCZ1, are decreased in globozoospermic sperm. Bioinformatics analysis indicates the Dpy19l2-deficient sperm presented molecular defects in acrosome, chromatin, sperm-egg interaction, and fertilization. The present study is the first to analyze total globozoospermia with Dpy19l2 deletion using high-throughput proteomics. This study may provide insights into the mechanism of globozoospermia.

Altered three-dimensional organization of sperm genome in DPY19L2-deficient globozoospermic patients.

To explore the three-dimensional (3D) organization of sperm genome in DPY19L2-deficient globozoospermic patients speculating a link between DPY19L2 and genome organization of sperm nucleus. This is a study of chromatin organization in DPY19L2-deficient globozoospermic patients and healthy donors using three-dimensional fluorescence in situ hybridization (3D-FISH) combined with confocal laser scanning microscopy followed by 3D image analysis. The 3D structures of sperm nuclei, chromocenter, telomeric regions and chromosome territories (CTs), were reconstructed using IMARIS software, and the relative radial position for each individual signal was calculated. Statistical analysis used a non-parametric Mann-Whitney test was appropriate with significance at p <0.05. DPY19L2-deficient globozoospermic patients display impaired sperm chromocenter organization resulting in an increased number of chromocenters (5.4 vs 3.5; p< 0.0001). Moreover, radial positions of telomeres are modified with a more central position in globozoospermic nuclei. 3D-FISH analysis of five chromosome territories (CTs) (X, Y, 7, 17, 18) showed that DPY19L2-deficient globozoospermic sperm nuclei display altered spatial organization of CT X, CT 7 and CT 18. Our findings strengthen the hypothesis that DPY19L2 might be considered as a LINC-like protein having a crucial role in the organization of nuclear chromatin in sperm nucleus through its interaction with nuclear lamina. Our results might also explain defective embryonic development after intracytoplasmic sperm injection (ICSI) performed with DPY19L2-deficient globozoospermic sperm.

Ultrastructural Morphology of Sperm from Human Globozoospermia.

Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia.

Dpy19l2-deficient globozoospermic sperm display altered genome packaging and DNA damage that compromises the initiation of embryo development.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia may be associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in the companion article that oocyte activation (OA) with globozoospermic sperm is very poor and due to the absence of phospholipase Cζ; therefore artificial OA (AOA) was used to bypass defective OA. Herein, we evaluated the developmental potential of embryos generated by ICSI + AOA in mice. We demonstrate that although OA was fully rescued, preimplantation development was impaired when using globozoospermic sperm. In human, a small number of embryos could be generated with sperm from DPY19L2-deleted patients in the absence of AOA and these embryos also showed a poor developmental potential. In conclusion, we show that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Most of the DNA breaks were already present when the sperm reached the epididymis, indicating that they occurred inside the testis. This result thus suggests that testicular sperm extraction in Dpy19l2-dependent globozoospermia is not recommended. These defects may largely explain the poor embryonic development of most mouse and human embryos obtained with globozoospermic sperm.

Subcellular localization of phospholipase Cζ in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospholipase C, zeta (PLCζ), the sperm factor thought to induce the Ca(2+) oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCζ in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCζ and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCζ is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca(2+) oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCζ in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equatorial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCζ in globozoospermic sperm is thus consistent and reinforces the role of PLCζ as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

Motile sperm organelle morphology evaluation-selected globozoospermic human sperm with an acrosomal bud exhibits novel patterns and higher levels of phospholipase C zeta

Does motile sperm organelle morphology examination (MSOME) affect levels and localization patterns of the oocyte activation factor phospholipase C zeta (PLCζ) in globozoospermic sperm with and without an acrosomal bud? MSOME identified round-headed globozoospermic sperm with increased levels of PLCζ relative to sperm from the same sample that did not undergo MSOME, and identified novel patterns of PLCζ localization in sperm exhibiting an acrosomal bud. Absence or reduction in the level of PLCζ in the sperm head, abnormal localization patterning, or defective functional ability as a result of PLCζ gene mutation, have been linked to certain types of human male factor infertility in which oocyte activation is deficient. It has been determined that a subpopulation of sperm (1%) from a patient exhibiting 100% globozoospermia presented with an acrosome bud upon MSOME. A cycle of intracytoplasmic morphologically selected sperm injection, carried out with sperm exhibiting an acrosomal bud led to pregnancy and birth of a healthy baby boy, without the use of assisted oocyte activation (AOA). Immunofluorescent analysis of PLCζ in globozoospermic sperm from three patients, before and after MSOME. Quantitative immunofluorescence was used to investigate PLCζ levels and localization patterns in individual sperm (n = 1 patient) identified by MSOME and isolated by micromanipulation, and presenting with and without the acrosomal bud. A secondary aim was to investigate levels and localization patterns of PLCζ in sperm before and after MSOME from two other globozoospermic men. Non-globozoospermic control sperm exhibited characteristic localization patterns of PLCζ immunofluorescence. Completely round-headed globozoospermic sperm from patients 1-3 were either devoid of PLCζ immunofluorescence, or exhibited an abnormal, punctate, pattern of PLCζ localization. PLCζ immunofluorescence in sperm exhibiting an acrosomal bud was observed in the midpiece with varying fluorescent intensity and was detected in 28.5% of such sperm. The majority of sperm with an acrosomal bud (43.0%) exhibited punctate patterns of PLCζ localization within the sperm head. A further 28.5% of sperm exhibited PLCζ in both the head and the midpiece. Total levels of PLCζ, and the proportions of sperm exhibiting PLCζ immunoreactivity, showed significant variance (P ≤ 0.05) amongst control [45.8 arbitrary units (a.u.) and 95.7%, respectively], non-MSOME-selected (25.9 a.u. and 46.1%, respectively) and MSOME-selected globozoospermic sperm (33.4 a.u. and 65.0%, respectively). Total levels of PLCζ immunofluorescence, and proportions of sperm exhibiting PLCζ immunoreactivity, in control sperm was significantly higher (P≤ 0.05) compared with non-MSOME-selected sperm, but not significantly different from MSOME-selected sperm. The low numbers of sperm analysed may not be ideal for conclusive statistical analysis. Evaluation of the effects of MSOME on morphologically normal sperm would confirm conclusions. The present findings provide hope for the future treatment of globozoospermia without the need for AOA, and provide further evidence for the clinical application of PLCζ as a therapeutic and prognostic tool. The research described herein was funded by the Nuffield Department of Obstetrics and Gynaecology, University of Oxford. The authors report no conflict of interest.

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation

To present the effectiveness of intracytoplasmic sperm injection (ICSI) using globozoospermic sperm and assisted oocyte activation by electrical stimulation. A case report. A private IVF center in Japan. A man with globozoospermia. Acridine orange (AO) test, mouse oocyte activation test, and ICSI with electrical oocyte activation. Fertilization, pregnancy, and live birth. In the first ICSI attempt, neither of the two injected oocytes fertilized. Staining of the patient's sperm with AO showed that only 2.9% of the sperm emitted a green fluorescence at the characteristic round head (sperm with native DNA content). The mouse oocyte activation test using the roundheaded sperm showed that the normal fertilization rate was 78.9% when SrCl(2) was used for assisted oocyte activation; however, it was 6.0% without assisted oocyte activation. We confirmed that the sperm had defective ability to activate oocytes. In the second ICSI attempt, human oocytes were activated electrically with use of a single square direct current pulse after microinjection. All the seven injected oocytes fertilized normally, and two eight-cell embryos were transferred on day 3. Clinical pregnancy was confirmed, and a healthy girl weighing 2362 g was delivered at 37 weeks of gestation by cesarean section. This is the first successful outcome of ICSI using globozoospermic sperm and electrical oocyte activation. The electroactivation obviates the need for the use of potentially harmful drugs for activation.

Fertilisation and pregnancy outcome after ICSI in globozoospermic patients without assisted oocyte activation

The successful outcome of intracytoplasmic sperm injection (ICSI) with globozoospermic sperm and non-activated oocytes is reported. Three couples underwent ICSI treatment and two of the patients were siblings. Forty-four non-activated oocytes were injected, 26 oocytes fertilised normally and 17 good quality embryos were obtained. Six embryo transfers were carried out, three with fresh embryos and three with frozen-thawed embryos. Three pregnancies resulted from the fresh embryo transfers and additionally two pregnancies were obtained after the transfer of frozen-thawed embryos. Two healthy babies were born. One twin pregnancy is ongoing. Our case reports demonstrate that in some ICSI attempts undertaken with globozoospermic sperm cells from two of our patients, high fertilisation rates, pregnancies and live births can be achieved, without artificially activated oocytes. Our data also suggest that in some cases, round-headed spermatozoa lack the capacity to activate the oocyte. Therefore, it cannot be excluded that artificial oocyte activation could be of help in globozoospermic patients with complete fertilisation failure.