Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos.

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 µL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

A single post-ovulatory dose of ulipristal acetate impairs post-fertilization events in mice.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator used for emergency contraception that has proven to be highly effective in preventing pregnancy when taken up to 120 h after unprotected sexual intercourse. Even though it may act mainly by delaying or inhibiting ovulation, additional effects of UPA on post-fertilization events cannot be excluded. Therefore, the aim of this study was to determine whether a single post-ovulatory dose of UPA could prevent pregnancy using the mouse as a pre-clinical model. Mated females received a single dose of UPA (40 mg/kg) on Day E1.5 or E2.5 (E0.5: copulatory plug detection) and post-fertilization events were evaluated. Our studies revealed that UPA administration produced a significant decrease in the number of conceptuses compared to control. Moreover, UPA-treated females exhibited a lower number of early implantation sites on Day E5.5, despite normal in vivo embryo development and transport to the uterus at E3.5. Administration of UPA produced histological and functional alterations in the uterine horns, i.e., a dyssynchronous growth between endometrial glands and stroma, with non-physiological combination of both fractions compared to controls, and a completely impaired ability to respond to an artificial decidualization stimulus. Altogether, our results show that the administration of a single post-ovulatory dose of UPA impairs mouse pregnancy probably due to an effect on embryo-uterine interaction, supporting additional effects of the drug on post-fertilization events. Although these studies cannot be performed with human samples, our results with the mouse model provide new insights into the mechanism of action of UPA as an emergency contraception method.