The effect of incubation time and temperature on sperm motility, human sperm DNA and assisted reproductive technologies (ART) outcome

Van Zyl, Estee Alwelien (2015-03)

Thesis (MMed)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: In all Assisted Reproductive Technologies (ART) procedures the semen sample is handled, processed, prepared and manipulated before use in the fertilization process. During these incubation times, the sperm cells are exposed to factors that may inflict damage to the sperm structure and DNA integrity, impair its functional abilities and subsequently lead to fertilization failure and poor ART outcome. Two of the very basic, but important factors that may have an impact on the sperm quality are time and temperature exposure. The primary objective of this study was to prospectively determine the effect of different incubation times and temperatures on motility and the DNA profile of the spermatozoa. Non-processed (n=36) and processed (n=33) semen samples were incubated for different time intervals (before: 20, 40, 60 minutes; after: 30, 60, 90 minutes) and at different temperatures (room temperature [RT] and 37°C). After incubation, sperm parameters were assessed, the CMA3 assay was applied to determine chromatin maturity and compaction and the TUNEL assay to assess the level of DNA fragmentation. The results showed that in the non-processed group, incubation led to a time-dependent, significant decline in the motility. The highest motility was seen at 20 minutes (37°C) and motility declined in a time-dependent manner. Incubation time and temperature did not affect the CMA3 and TUNEL values. Incubation of the processed sample led to a significant time-dependent decrease in the motility; 90 minutes (RT) had the lowest motility. The CMA3 and TUNEL values between the different incubation groups did not differ significantly. The secondary objective was to retrospectively investigate the effect of sperm incubation time after preparation on ART outcome. A total of 901 patient ART cycles (January 2010- December 2012) were included. Fertilization rates, embryo quality and pregnancy rates were examined. The results showed that the sperm incubation time before insemination between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) differed significantly and the incubation time had a significant negative effect on the fertilization rates in IVF, but not in ICSI. Longer incubation times led to an unexpected significant improvement in the quality of day 2 embryos and were significantly associated with pregnancy failure in IVF and ICSI. These combined findings suggest that non-processed semen samples can be incubated at RT or 37°C, but for no longer than 40 minutes and, for IVF, processed samples should not be incubated for longer than 60 minutes at RT or 37°C. The ICSI sample should not be incubated for more than 60 minutes although longer incubation times do not seem to influence the results for IVF. It can therefore also be concluded that sperm incubation time before insemination should be closely monitored, especially in IVF cycles.

AFRIKAANSE OPSOMMING: In kunsmatige voortplantingstegnieke (ART) word die semen-monster geprosesseer, voorberei en gemanipuleer voordat dit vir die bevrugtingsproses gebruik word. Terwyl die monster geïnkubeer word, word die spermselle blootgestel aan verskeie faktore wat die struktuur van die sperm, die DNS integriteit en die sperm se funksionele vermoë negatief kan beïnvloed. Dit kan lei tot swak bevrugting, embriokwaliteit en swangerskapsyfers. Twee basiese, maar belangrike, faktore wat die spermkwaliteit negatief kan beinvloed is die duur van inkubasie en die temperatuur waarby die spermselle geïnkubeer word. Die primêre doel van die huidige studie was om prospektief te ondersoek wat die effek van verskillende inkubasietye en temperature op die motiliteit en DNA profiel van die sperm het. Monsters is voor en na spermvoorbereiding vir verskillende tydsintervalle (voor: 20, 40, 60 minute; na: 30, 60, 90 minute) en verskillende temperature (kamertemperatuur [KT] en 37°C) geïnkubeer. Na elke inkubasie is ’n spermanalise, ’n CMA3- en ’n TUNEL toets gedoen. Die CMA3 toets bepaal die chromatienmaturiteit en -kompaksie en die TUNEL toets vir die vlak van DNS fragmentasie. Die resultate het getoon dat daar in die voor voorbereiding groep ’n beduidende verskil in motiliteit tussen die verskillende inkubasiegroepe was. Die hoogste motiliteit is in die 20 minute/37°C groep gevind. Die motiliteit het oor tyd afgeneem. Die tyd en temperatuur van inkubasie het nie ’n beduidende effek op die CMA3 en TUNEL uitslae gehad nie. Inkubasie nadat die semen voorberei was het weereens tot ’n beduidende verskil in motilieit tussen die groepe gelei. Die laagste motiliteit is waargeneem by 90 minute/KT. Geen beduidende verskil is tussen die inkubasiegroepe vir CMA3 en TUNEL gevind nie. Die sekondêre doel van die studie was om retrospektief te ondersoek wat die effek van sperminkubasietyd na spermvoorbereiding op die bevrugting, embriokwaliteit en swangerskapsyfers is. 901 pasiëntsiklusse is in die studie ingesluit (Januarie 2010 tot Desember 2012). Die resultate het aangedui dat die inkubasietye van die intrasitoplasmatiese inspuiting (ICSI) en in vitro bevrugting (IVB) beduidend van mekaar verskil het. Langer inkubasietye het ’n beduidende negatiewe effek op die bevrugtinguitslae van IVB siklusse gehad, maar geen effek op ICSI siklusse gehad nie. Langer inkubasietye het ook tot ’n onverwagte verhoging in die kwaliteit van dag 2 embrios gelei en was verder beduidend geassosieer met negatiewe swangerskapuitkoms. Hierdie gesamentlike bevindinge dui aan dat semenmonsters voor voorbereiding by KT of 37°C geïnkubeer kan word, maar nie vir langer as 40 minute nie. Na semenvoorbereiding, behoort die IVB semenmonster vir nie langer as 60 minute voor inseminasie geïnkubeer te word nie (KT of 37°C). Die ICSI semenmonster moet verkieslik binne 60 minute na voorbereiding gebruik word, maar dit wil voorkom asof die tyd hier nie so ’n groot rol speel nie. Daar kan verder afgelei word dat sperminkubasietye voor die gebruik vir inseminasie baie goed gemonitor moet word – veral in IVB siklusse.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96793
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