128 Effect of Perifollicular Blood Flow on the Quality of Oocytes Collected During Repeated Opu Sessions

Abstract

Approximately 20% of the cumulus-oocyte complexes (COC) collected from living animals during repeated ovum pick-up (OPU) sessions develop to the blastocyst stage. An increase in the blood supply to individual follicles appears to be associated with follicular growth rates, while a reduction seems to be closely related to follicular atresia (Acosta TJ et al. 2003; Reproduction, 125, 759-767). Recently, it has been shown that differences in perifollicular blood flow during repeated OPU sessions once weekly were predictive of oocyte competence. The purpose of this study was to determine whether qualitative perifollicular blood flow changes affect the quality of oocytes collected during repeated OPU sessions once or twice weekly as well as the quality of the resulting blastocysts. Lactating Holstein cows (n = 20) were used as oocyte donors. After dominant follicle removal, OPU was performed twice (group 1, for 3 weeks) or once (group 2, for six weeks) weekly employing a 7.5 MHz transducer (GE 8C-RS) of an ultrasound scanner (GE Logiq Book). Doppler characteristics were recorded by transvaginal ultrasonography just before COC collection using the color flow imaging. Because of technical limitations for measurement of blood flow in small individual follicles, only the presence or absence of blood flow was assessed for each follicle. When a clearly visible blue or red spot (blood flow) was detected in the follicle wall, it was considered a follicle with detectable blood flow. Follicles with or without detectable blood flow from each individual cow were aspirated separately. After morphological classification of COC, standard protocols for IVP were used for blastocyst production. For mRNA analysis, denuded COC and blastocysts were frozen at -80°C to analyze the relative transcript abundance using RT-qPCR. The transcripts studiedplay important roles during oocyte and embryo development [DNA methyltransferase 1a, 1b, 3a (DNMT1a, DNMT1b, DNMT3a); histone deacetylase 2 (HDAC2); growth differentiation factor 9 (GDF9); bone morphogenetic protein 15 (BMP15); maternal effect gene zygotic arrest (ZAR); heat shock protein 70.1 (HSP); glucose transporter1, 3 (GLUT1, GLUT3); glucose-6-phosphate dehydrogenase (G6PD); and desmocollin II (DCII)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. The relative abundances of ZAR, BMP15, GDF9, DNMT1a, DNMT3a, and HDAC2 transcripts were significantly upregulated in oocytes stemming from OPU sessions twice weekly, whereas qualitative blood flow changes did not influence the mRNA abundance. At the blastocyst stage, G6PD mRNA was upregulated in blastocysts generated from oocytes collected in OPU sessions twice weekly. These results show that the time interval between the individual OPU sessions had an effect on the quality of oocyte and embryos at the molecular level, whereas differences in the perifollicular blood flow did not. Ruthe Research Farm, Germany for providing the animals; Masterrind GmbH, Germany for donation of the semen, and the HW Schaumann Stiftung for financial support.

In vitro culture systems: how far are we from optimal conditions?

“…Blood flow determinations of individual preovulatory follicles prior to follicular aspiration for IVF therapy provide an important insight on the intrafollicular environment and may predict the developmental competence of the corresponding oocyte (Coulam et al, 1999;Huey et al, 1999). In cattle, it has been shown that the time interval between the individual OPU sessions had an effect on the quality of oocyte and embryos at the molecular level, whereas differences in the perifollicular blood flow did not (Hanstedt et al, 2010). An increase in the blood supply to individual follicles appears to be associated with follicular growth rates, while a reduction seems to be closely related to follicular atresia (Acosta et al, 2003;Acosta 2007).…”