Accidental exposure of oocyte/cumulus complex to endometriotic fluid is not uncommon during oocyte retrieval. Unfortunately, there is scanty and contradictory data on this subject to counsel patients or guide us in our clinical practice. To avoid the ethical and practical problem of dealing with human oocytes and embryos, mice were used as the animal model to explore the effects of endometriotic fluid exposure on oocyte fertilization and subsequent embryo development. The mouse embryo culture system has been used for many years as a tool to monitor culture conditions for human in vitro fertilization . Moreover, it is useful as a bioassay for evaluating the potential toxicity of environmental contaminants on human fertilization and early embryo development [5, 6]. An exposure time of five minutes was chosen in this study because most oocytes/cumulus complexes would have been removed from the contaminated follicular fluid during oocyte retrieval within this period. Our experimental model could be considered as an extreme condition, because in real-life situations oocyte/cumulus complexes are exposed to endometriotic fluid mixed with follicular fluid/flushing medium for a much shorter duration. As there was no significant decrease in fertilization rate of oocytes exposed to endometriotic fluid, the effect of exposure to serial dilution of the fluid or shorter exposure time was not studied further.
In this study, oocytes/cumulus complexes were exposed to endometriotic fluid to assess its effect on fertilization and subsequent embryo development. In the control group, they were exposed to fertilization medium before insemination in the same medium. We included two more comparison groups as exposure of oocytes/cumulus complexes to another fluid/medium with different basic compositions, pH and osmolality from fertilization medium (pH 7.5-7.8, osmolality 285–295 mOsm/kg) could induce stress to the oocytes, and had an effect by itself on subsequent embryo development. In one group, oocytes/cumulus complexes were exposed to PBS, which had a pH of 7.4 ± 0.1 and an osmolality of 282–288 mOsm/kg. In another comparison group, we exposed them to pooled sera from normal subjects without endometrioma. The rationale was that endometriotic fluid was a collection of old blood. Its composition should, therefore, be similar to serum with less water and the presence of degenerated red blood cells, inflammatory cells, and other possible toxic products to oocytes/early embryos. We treated the pooled sera the same way as endometriotic fluid, in terms of storage, thawing, and exposure to oocyte/cumulus complexes. If endometriotic fluid did contain some toxic factors, we expected that oocyte/cumulus complexes exposure would show significant decrease in fertilization and/or cleavage and/or blastocyst formation rate compared to those exposed to pooled sera from subjects without endometrioma. The fact that there was no difference in the developmental competence of oocytes that were exposed to endometrioma (group 1) or pooled sera of subjects without endometrioma (group 2), suggested that there was no direct influences of the endometriotic content per se. The significant decrease in the proportion of hatching/hatched blastocysts in these two groups, compared to those exposed to fertilization medium (control) and PBS (group 3), could be due to nonspecific effects common to both the endometriotic fluid and pooled sera, such as suboptimal pH and osmolality of the fluid [7, 8] as both of them were not equilibrated with 6% CO2 or adjusted for osmolality before use.
The fertilization rates of 52.1% and 51.6% in oocytes/cumulus complexes that were previously exposed to PBS and fertilization medium (control) were compatible with a previous report by Vergara et al. , for this strain of mouse (52%), using a similar superovulation protocol and culture conditions. Our blastocyst formation rate of 50-60% and the mean (±SD) number of cells in our blastocysts (55–64 cells) was also in line with other previous reports for ICR mouse blastocysts that were produced in vitro (blastulation rate of 27%-54% and blastocyst cell count of 22–84) [9–12].
Our study (n = 200 oocytes/group) was designed to detect a 25% decrease in fertilization rate of oocytes after exposure to endometriotic fluid, with a power of 80% and a type-I error of 5% (two-tailed). A smaller decrease in fertilization rate of 10% or less, as suggested by Suwajanakorn et al., could have gone unnoticed. In agreement with Suwajanakorn et al. and Khamsi et al., we did not observe a detrimental effect of endometriotic fluid on cleavage rate of the zygotes or the formation of good-quality embryos during our daily observation of embryo development. We also found no significant difference in the blastulation rate between the exposed and non-exposed oocytes, which was not reported in the two previous studies. However, the proportion of hatching and hatched blastocysts was significantly lower in the fertilized oocytes that were previously exposed to endometriotic fluid. It was reassuring that hatching blastocysts from all experimental groups and controls had no significant difference in the number of inner cell mass or trophectoderm or total cells. However, the implantation and pregnancy potential of these blastocysts could not be determined, as we did not transfer them into the uterus.