Administration of ethiodized poppy seed oil-based contrast agent into the uterus enhances fertilization rate in mice inseminated with low sperm numbers.

study questionDoes the administration of an oil-based iodinated contrast medium (OSCM) in the mouse uterus have a positive effect on fertility similarly to what is observed in women following hysterosalpingography (HSG)? SUMMARY ANSWER: Following IUI of a small number of sperm (mimicking oligozoospermia), fertilization and birth rates were improved in female mice who had previously received an intrauterine administration of an OSCM confirming a pro-fertility effect. WHAT IS KNOWN ALREADY: HSG is a common diagnostic procedure used in infertile or subfertile women, primarily to assess the patency of fallopian tubes. It consists in injecting an iodinated contrast medium into the uterus to enable the imaging of the female reproductive tract by radiography. In the absence of obstruction, the contrast medium fills the uterus and transits through the fallopian tubes before overflowing in the peritoneal cavity. Several studies reported that in the few months following HSG, women injected with an OSCM (ethyl esters of iodinated fatty acids of poppy seed oil) had an enhanced pregnancy rate (either spontaneous or following IUI) compared to those administered with a water-based contrast medium. This suggests that OSCM administration in the female reproductive tract could improve fertility. However, the physiological mechanisms underlying this potential effect remain unknown. STUDY DESIGN, SIZE, DURATION: OSCM or PBS (phosphate buffered saline, control) were administered into the uteri of B6D2 female mice (5-9 weeks old, total n = 73). Two weeks later, intrauterine insemination was performed with mouse epididymis sperm from B6D2 male mice (8-16 weeks old). Females were euthanized and fertilized oocytes collected and incubated for 4 days up to the blastocyst stage (n = 52). Alternatively, females were left until delivery (n = 21). PARTICIPANTS/MATERIALS, SETTING,

methodsFemale nulliparous mice were administered either with PBS or OSCM directly into the uterus. After 2 weeks, females were inseminated with a controlled number of sperm. The resulting fertilized/unfertilized oocytes were collected and counted the following day to calculate the fertilization rates and then further incubated in vitro to follow the development to the blastocyst stage. In addition, females were mated with vasectomized males to allow implantation, subsequent pregnancy and birth. MAIN RESULTS AND THE ROLE OF CHANCE: First, females were inseminated with a high number of sperm (5 × 10e5 spermatozoa). Comparable fertilization rates (67.0 ± 5.4% and 60.0 ± 8.0%, P > 0.05) were observed from oocytes originating from OSCM and PBS-administered mice, respectively. This showed that when inseminating with a high number of sperm, uterine OSCM administration had no deleterious effect but did not improve fertilization. We postulated that the potential beneficial effect of OSCM administration might be masked by the optimal reproductive system of young mice. It was therefore decided to test the effect of OSCM in suboptimal reproductive conditions such as those observed when sperm concentration is low (oligozoospermia). As could be expected, fertilization rate decreased proportionally to the reduction of inseminated sperm suggesting that it is a relevant model for oligozoospermia and to mimic subfertility. When using a suboptimal number of spermatozoa (15 × 10e3), the fertilization rate was significantly increased in OSCM administered mice compared to controls (29.9 ± 4.8% and 12.6 ± 3.0%, P < 0.05, respectively). Moreover, OSCM administered females also gave birth to a higher number of pups than controls (3.7 ± 1.7 and 0.9 ± 0.9, P < 0.05). LARGE SCALE DATA: No large scale data were produced in this study. LIMITATIONS, REASONS FOR CAUTION: The reason for studying the effects of oil soluble iodinated contrast media on mouse fertility is based on the results from several clinical studies in humans. Those reports included women of different age and suffering from different types of infertility or subfertility. Moreover, the timing of their pregnancies after OSCM treatment also occurred at different times. Our mouse model and experiments do not include such heterogeneity as the inclusion of variables related to age, time of pregnancy and fertility deficiencies, which would demand a much larger number of mice, contrary to the 3R principles (Replacement, Reduction and Refinement of animal experimentation). For the same reason, our results are based upon mice treated with OSCM in contrast to mice treated with PBS, while human clinical reports may include alternatives to OSCM. Moreover, it is important to note that mouse and human reproductive systems differ at several levels such as morphology, hormonal influences, oestrus, menstrual cycles and pregnancy, raising the possibility that OSCM action may differ across species. In our mouse model, OSCM does not cross the uterotubal junction in the mice during administration, restricting it to the uterus, thus excluding any possible effects on the oviducts and peritoneal cavity, which are reached by OSCM in humans. Additionally, as the mouse reproductive cycle is much faster than that of women, it is likely that the amount of OSCM remaining in the mouse uterus after 2 weeks differs from what is left in women after several months. WIDER IMPLICATIONS OF THE

findingsIn our mouse model, OSCM administration in the uterus increased the number of embryos and pups obtained when inseminated with low sperm numbers. This suggests that the increased fertilization rate is most probably due to an effect on the uterus itself or on the sperm which go through it before reaching the fertilization site. In the mouse, the presence of OSCM in the oviduct and inside the peritoneal cavity is not necessary to observe a beneficial effect of the contrast agent. These initial results demonstrate that the mouse model could be used to further study the OSCM mechanisms in the uterus. Most importantly, our results confirm a beneficial effect of OSCM on fertility and suggest that it could help couples with fertility issues, improving fertilization and pregnancy rates following IUI with substandard sperm samples. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Guerbet Group, INSERM, CNRS, Université Grenoble Alpes and the French Agence Nationale pour la Recherche (ANR) grant "LIPIOFERT" (ANR-23-CE18-0039) to C.L. Guerbet Group is producer and distributor of Lipiodol®, provided by Guerbet free of charge for this study. The study was partly financed by Guerbet and M.R. and P.Ro. are Guerbet's employees. Data were produced and analysed independently by academic researchers from the GETI's laboratory E.L., C.A., P.Ra., and C.L. E.L. was temporarily employed by Guerbet from 1 December 2022 to 31 May 2023.