Kinetics of human male pronuclear development in a heterologous ICSI model.

PURPOSE: To evaluate human sperm nuclear chromatin decondensation in a heterologous ICSI system using hamster ova injected with human sperm. MATERIALS AND METHODS: Frozen hamster oocytes were injected with Triton X-100 treated sperm and fixed at different time points post ICSI. Oocytes injected with non-treated sperm served as controls. Male pronuclear decondensation was evaluated after staining with DAPI. RESULTS: Sperm cells with partially destroyed membranes and depletion of the acrosome decondense more rapidly and to a greater extent than membrane/acrosome intact cells. Marked variability in pronuclear size was observed for any time point post ICSI, which most probably reflects the heterogeneity in the mature human sperm population. CONCLUSION: Remodeling of male gamete nuclei in this heterologous ICSI mimics events that occur during natural fertilization in humans and therefore this approach may be used for studies of human sperm chromosomes transformations.

Neurotensin stimulates the sperm acrosome reaction and reduces percentages of fertilization in vitro.

OBJECTIVE: To determine the impact of neurotensin (NTS), a naturally occurring peptide, on the function of human and nonhuman primate sperm. DESIGN: Experimental study. SETTING: University-based research laboratory. PATIENT(S)/ANIMAL(S): Consenting normozoospermic human donors and cynomolgus macaques. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm acrosome status was assessed. Computer-assisted semen analysis assessed sperm motility, progression, and velocity. Immunocytochemistry and receptor selective agonists were used to identify specific NTS receptors on sperm. Monkey oocytes were obtained after ovarian stimulation, and NTS-treated monkey sperm were used for in vitro fertilization. RESULTS: Neurotensin treatment of human sperm stimulated the acrosome reaction in both a dose-dependent (0.1-10 µmol/L) and time-dependent (5-30 minutes) manner. Neurotensin treatment did not alter sperm motility or progression. Both a general NTS receptor antagonist (SR142948) and a NTSR1 selective antagonist (SR48692) reduced the ability of NTS to stimulate the acrosome reaction. The neurotensin receptor NTSR1, but not NTSR2 or SORT1, was detected in monkey sperm using immunostaining. Neurotensin treatment also compromised the ability of sperm to fertilize an oocyte. Percentage of fertilization with untreated monkey sperm and monkey oocytes was 72%. Sperm pre-treated with NTS yielded a significantly lower fertilization rate of 18%. CONCLUSION(S): Neurotensin effectively stimulates the acrosome reaction in human and monkey sperm. Neurotensin produced by the oviduct or cumulus cells may promote natural fertilization. Pretreatment of sperm with NTS significantly reduces fertilization. Exposure of sperm to NTS prior to reaching the oviduct has the potential for contraceptive development. Identification of NTSR1 as the mediator of NTS action provides a specific target for future studies.

Positioning of chromosomes in human spermatozoa is determined by ordered centromere arrangement.

The intranuclear positioning of chromosomes (CHRs) is a well-documented fact; however, mechanisms directing such ordering remain unclear. Unlike somatic cells, human spermatozoa contain distinct spatial markers and have asymmetric nuclei which make them a unique model for localizing CHR territories and matching peri-centromere domains. In this study, we established statistically preferential longitudinal and lateral positioning for eight CHRs. Both parameters demonstrated a correlation with the CHR gene densities but not with their sizes. Intranuclear non-random positioning of the CHRs was found to be driven by a specific linear order of centromeres physically interconnected in continuous arrays. In diploid spermatozoa, linear order of peri-centromeres was identical in two genome sets and essentially matched the arrangement established for haploid cells. We propose that the non-random longitudinal order of CHRs in human spermatozoa is generated during meiotic stages of spermatogenesis. The specific arrangement of sperm CHRs may serve as an epigenetic basis for differential transcription/replication and direct spatial CHR organization during early embryogenesis.

Protamine withdrawal from human sperm nuclei following heterologous ICSI into hamster oocytes.

During late stages of spermatogenesis in mammals, most histones bound to DNA are replaced by protamines (PRM), which results in formation of supercondensed and genetically inert sperm chromatin. At fertilization, mature spermatozoon penetrates oocyte and chromatin is remodeled "back" from nucleoprotamine to nucleohistone state. While being crucial for activation of male genome and ultimately for initiation of embryonic development, this process is poorly studied, especially in humans. Data on model animals concerning PRM to histones exchange post fertilization are few and contradictory. As direct experimentation with human embryos is impossible due to ethical, legal and technical reasons, we evaluate the timing and mode of PRM removal in a heterologous ICSI system using hamster ova injected with human sperm. Localization of human PRM 1 and 2 in hybrid zygotes was established using immunofluorescence. We observed a marked zygote to zygote variability in male pronuclei size for any time point post ICSI and demonstrated that PRM removal correlates with the developing pronuclei area rather than time after injection. Overall, the disappearance of protamines from sperm is rather rapid and most likely completed within 1 hr. We propose that the critical characteristic influencing PRM removal after heterologous fertilization is the intrinsic heterogeneity of the human sperm population. The same yet unexplored variance may be one of the reasons for canceled, delayed or aberrant early embryonic development during natural or artificial fertilization in humans.

Impact of transabdominal ultrasound guidance on performance and outcome of transcervical uterine embryo transfer.

PURPOSE: To determine the impact of transabdominal ultrasound guidance on embryo transfer during IVF therapy. METHODS: Retrospective analysis of 823 consecutive embryo transfers. Three hundred and sixty-seven procedures performed with transabdominal ultrasound guidance were compared to 456 cases performed with the "clinical touch" method. RESULTS: Ultrasound-guided embryo transfer yielded higher, but not statistically significant, clinical pregnancy (48% vs. 44%) and implantation rates (22% vs. 20%). The incidence of multiple pregnancies, ectopic and multiple pregnancy rates were similar. The frequency of negative factors typically associated with difficult transfers, such as requirement of use of tenaculum, and presence of blood or mucus in the catheter tip, were significantly lower in the ultrasound-guided group in comparison with the clinical touch group. Ultrasound-guided embryo transfer was associated with a significantly increased easiness of transfer performance; 95% of the transfers were rated as very easy in the ultrasound-guidance group compared to 87% in the clinical touch group. The use of a soft pass catheter was the only variable independently and significantly associated with pregnancy success (odds ratio = 2.74). CONCLUSION(S): Ultrasound-guidance facilitates embryo transfer and in combination with the use of a soft catheter should be implemented to optimize embryo transfer results.