Delayed pronuclear appearance as an indication of compromised oocyte repair capacity

To associate the delayed formation of two-pronuclei (2PN) after ICSI with the integrity of the oocytes capability to cope and eventually overcome varying levels of genomic error. In a retrospective study, we investigated the impact of the degree of sperm DNA fragmentation in relation to oocyte aging. We gauged the efficiency of oocyte repair mechanisms in occasion of delayed pronuclear appearance and consequent syngamy was assessed. We identified couples undergoing ICSI treatment (n=152) that had sperm chromatin assessment by TUNEL on their ejaculates. TUNEL was performed by adjusting sperm concentration to 5 million, fixation with 4% PFA, exposure to mild permeabilizing solution, and incubated at 37°C with TUNEL reaction mixture. At least 500 spermatozoa were scored under fluorescent microscopy. Patients were categorized by DFI level and grouped according to maternal age. Each subdivision of DFI was evaluated in relation to clinical pregnancy outcome (presence of a fetal heartbeat). Standard fertilization is performed 16-18 hours after ICSI and delayed fertilization was conducted over 18 hours. To determine the significance of a delayed normal fertilization, arguably linked to an extensive processing and repair of the male genome following sperm nuclear unraveling, we assessed cohorts of oocytes that had at least one oocyte with a delayed appearance of the pronuclei in relation to a matched control. In a previous study, we established in 315 ICSI cycles which included 152 women with a mean age of 37.3±4yrs. In a cohort of women <35yo of age, their young oocytes were able to prevent a deleterious effect in spite of the increasing sperm DFI on the pregnancy rate. On the other hand, in women ≥35yo the pregnancy rate dropped to 5.6% when the DFI was just barely at the threshold of 20% (P<0.01), reflecting a compromised oocyte repair ability towards the male DNA. In 153 couples treated in 362 ICSI cycles, a delayed bipronuclear fertilization appeared in 2.4% (362/14,859) that included men (40.9±3yrs) with concentration of 49.3million, motility of 46.7%, and morphology of 2.8%. Also in this cohort when we looked at the <35yo women, a delayed fertilization did not affect pregnancy rate and characteristics. In the group of women ≥35yrs the fertilization fertilization, clinical pregnancy, and embryo implantation (P<0.01) were all compromised when compared to an age-matched control (n=14,859). This study confirms the ability of ICSI to overcome spermatozoa with a compromised paternal genome. This phenomenon appear linked to the ability of the oocyte to process and repair male DNA very efficiently for women of a younger age and clearly impaired in a population of ≥35 years old. The occurrence of this repair mechanism may be visualized by a delayed appearance of the biparental pronuclei that when present in older oocytes is linked to a compromised clinical outcome.