Zygote Diameter and Total Cytoplasmic Volume as Useful Predictive Tools of Blastocyst Quality.

Introduction According to the Embryo Protection Act, the selection of embryos with the greatest potential for successful implantation in Germany must be performed in the pronucleus stage. The main aim of this study was to identify morphokinetic parameters that could serve as noninvasive biomarkers of blastocyst quality in countries with restrictive reproductive medicine laws. Materials and Methods The sample comprised 191 embryos from 40 patients undergoing antagonist cycles for intracytoplasmic sperm injection. Blastocysts were cultured in an EmbryoScope chamber and video records were validated to determine the post-injection timing of various developmental stages, cleavage stages, and blastocyst formation. The Gardner and Schoolcraft scoring system was used to characterize blastocyst quality. Results Morphokinetic data showed that the zygote diameter and total cytoplasmic volume were significantly different between good and poor blastocysts quality groups, where zygotes, which formed better blastocyst quality, had smaller diameter and smaller total cytoplasmic volume. Zygotes with more rapid pronuclear disappearance developed in better-quality blastocysts. Differences between good- and poor-quality blastocysts were also observed for late-stage parameters and for the spatial arrangement of blastomere where tetrahedral embryos more frequently forming good-quality blastocyst compare to the non-tetrahedral. Conclusions The study findings could be used to enhance embryo selection, especially in countries with strict Embryo Law Regulations. Further studies, including those in which the implantation potential and pregnancy rate are considered, are warranted to confirm these preliminary results.

The impact of anti-Müllerian hormone (AMH) on multiple pronuclei (PN) presence and oocyte maturity in ICSI treatments.

This prospective study was designed to investigate whether anti-Müllerian hormone (AMH) levels are associated with the presence of multiple pronuclei in zygotes as well as with the ovarian response, fertilization rate and pregnancy outcome in ICSI cycles. A total of 413 patients undergoing ICSI cycles were included in the study. The assessment included 3084 MII oocytes. Serum AMH measurements were performed at the first initial presence of the patient. The outcome measures were the presence of multiple pronuclei (PN), a number of retrieved oocytes, number of mature/immature oocytes, fertilization rate and clinical pregnancy. Obtained results showed a statistically significant correlation between AMH levels and maternal age, the number of follicles, the number of cumulus-oocyte complexes, mature and immature oocyte, fertilization rate and pregnancy rate. Linear regression analysis showed that AMH significantly correlates with the presence of multiple pronuclei in the zygote. The further analysis confirmed that the number of zygotes with the presence of multiple pronuclei increased when AMH levels were higher. This is the first examination of the prognostic value of the serum AMH on the presence of multiple pronuclei in the zygote and our data in the preliminary study suggest that AMH levels could be used as a predictive marker.

Positive effect of progesterone on motility and velocity of fresh, vitrified without permeable cryoprotectants and frozen with permeable cryoprotectants human spermatozoa.

Steroid hormone progesterone has been found to play an important role in the migration of spermatozoa through the reproductive tract, as well as to induce hyperactive motility and increase sperm velocity. The aim of this study was to examine whether progesterone could induce beneficial effects in vitrified and slow-frozen spermatozoa. During the research process, 50 semen samples were divided into three treatment groups; noncryopreserved, slow-freezing and vitrification. After thawing and an incubation period of 2 hr to induce capacitation, semen samples from each treatment group were treated with 50 nM, 25 nM progesterone and a control solution for 30 min. Thereafter, the sperm suspensions were examined manually to assess the proportion of viable and motile spermatozoa, as well as using the CASA to evaluate the velocity parameters. The results indicated a higher proportion of progressively motile spermatozoa in vitrified teratozoospermic samples and improved velocity parameters in slow-frozen normozoospermic and teratozoospermic samples. The main conclusion of this research was that the used progesterone concentration of 50 nM was sufficient to significantly improve the motility of vitrified teratozoospermic samples and velocity parameters of cryopreserved sperm samples. The present findings might have important implications in determining ways of improving the current low rates of motility in cryopreserved spermatozoa.

Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress.

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O2- localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.