Preliminary observations on embryo morphokinetics and sperm DNA fragmentation assessed using the halo sperm kit

Abstract

Sperm DNA fragmentation has been suggested to be a contributor to male infertility and lower pregnancy outcomes. The degree to which DNA fragmentation influences outcomes in IVF/ICSI cycles is however still controversial. Integration of time-lapse microscopy (TLM) in to IVF labs allows more in depth analysis of embryo developmental kinetics than was previously possible with conventional microscopy. This study examines sperm DNA fragmentation and its relationship to embryo morphokinetics, multinucleation and cleavage anomalies. Pilot study looking at DNA fragmentation (DF) and embryo morphokinetics. This study encompassed 77 couples undergoing IVF. Sperm specimens were prepared for ICSI using density gradient centrifugation. Mature oocytes were fertilized by ICSI and immature oocytes were incubated overnight with sperm. Normally fertilized oocytes were cultured in the EmbryoScope time lapse chamber. Timing of specific events from the point of insemination was determined using time lapse (TL) imaging and expressed in hours. The following kinetic markers were assessed: time to syngamy (t-syn), time to 2c (t2), 3c (t3), 4c (t4), 5c (t5), 8c (t8), morula (Mor-t), start of blastulation (tSB), blastocyst (BL-t), and expanded blastocyst (EBL-t). Durations of the second (cc2) and third (cc3) cell cycles as well as time to complete synchronous divisions s2 and s3 were calculated. Embryos were also observed for presence or multinucleation and cleavage anomalies. Sperm DNA fragmentation testing was performed on an aliquot of the unused ICSI sperm preparation using the Halosperm kit (HaloTech/Spectrum Technologies). The kit enabled microscopic detection of sperm chromatins dispersion. Sperm were embedded in agarose coated slides and exposed to acid denaturation and a lysis buffer. Following alcohol dehydration and staining with DiffQuik, slides were examined to detect presence of halos surrounding sperm. Sperm cells with large/medium sized halos characteristic of dispersed DNA loops were considered to have intact DNA. Sperm with small, degraded or absent halos were considered to have fragmented DNA. Cases were divided according to % DF in ICSI specimens. Morphokinetic parameters were compared. P value of <0.05 were considered to be statistically significant. Only parameters that were significantly different are shown in the table.Tabled 1Morphokinetics and DNA fragmentationDNA Fragmentation <15%DNA Fragmentation>=15%P ValueTotal embryos63354Multinucleation47%69%<0.01Irregular chaotic division25%46%<0.001Morula formation79%66%<0.05Start of blastulation74%60%<0.05Early blastocyst formation66%48%<0.01Expanded blastocyst formation52%32%<0.01t439.6+ 0.342.2 + 1.2<0.01t4-t32.7 + 0.24.8 + 0.7<0.01t859.5 + 0.564.7 + 2.2<0.01 Open table in a new tab The Halosperm kit provided an easy inexpensive means to assay DNA fragmentation. Further study is needed on the association between sperm DNA fragmentation and embryo quality markers made possible thru time lapse imaging systems.