Vaginal Delivery of Concentrated Oxygen and Hyaluronic Acid Improves the Implantation Rate in Human IVF: A Proof of Concept Study

1. Introduction

Implantation failure following assisted reproductive techniques often remains an unresolved dilemma. Although embryo culture has improved notably over the past two decades there have been less advances in understanding endometrial receptivity. It has been estimated that embryo viability accounts for one third of implantation failures, while sub-optimal endometrial receptivity and altered embryo-endometrial dialogue are responsible for the remaining two thirds (1,2,3). Even following blastocyst selection using pre-implantation genetic diagnosis for ploidy, the implantation rate hovers around 40-60% (4). Embryo implantation in the human is a complex orchestration of complimentary tissues, involving a temporal sequence of dialogue and depends on an oxygen and nutrient rich uterine environment (5).

Since the classical work of Rock and Bartlett (6), describing the histological changes of the endometrium at the time of implantation, there have been many attempts to improve the efficiency of implantation. Protocols for the timing of transfer of in vitro human embryos revolve around monitoring the endothelial lining with ultrasound (7). A wide variety of pharmaceutical options to improve implantation rates exist and range from hormonal therapy, through cortisone to vasodilators. Specific attempts to identify the window of implantation have seen the introduction of the ERA test (Endometrial Receptivity Array) where several hundred molecular markers have been suggested to be indicative of a receptive endometrium (8). To date, the ERA test is considered to be of limited clinical use (9). Mechanical stimulation of the uterine lining by endometrial scratching has been used for decades in an attempt to improve the implantation rate (10), while much attention has been given to immune alterations and therapies in human endometrial receptivity (11). Attempts to improve endometrial receptivity by infiltration of platelet-rich plasma (PRP) has also recently been adopted (12), while the elasticity of the cervix has been considered as a marker for ease of transfer and improved implantation rates (13). Uterine contractility at the time of embryo transfer, cervical stenosis and indeed the adequacy of the embryo transfer technique may all lead to negative outcomes after assisted reproduction (14).

An equilibrated vaginal microbiome has been used as a marker to indicate a favourable implantation platform (15) and recently it has been shown that the vaginal biome may be significantly altered following erogation with high concentrations of oxygen and hyaluronic acid (16), with marked improvements in hydration of the vaginal epithelia. This minimally invasive technology is now widely used to treat a variety of vaginal and vulval pathologies, including lichens, atrophy, urethral pain, candidiasis and vaginal dryness (17,18,19).

More recently, it has been demonstrated that microbial contamination of the embryo transfer catheter with cervico -vaginal microorganisms, other than Lactobacilli, can significantly reduce pregnancy and live birth rates in an IVF programme (20).

The present study is a pilot, proof of concept, retrospective, observational case controlled study to determine whether high O₂ and hyaluronic acid applied vaginally in the two weeks preceding an embryo transfer may increase implantation rates. Frozen-thawed blastocysts were used in single embryo transfers in 377 patients from CFA-Italy, Naples, randomly assigned to a control group and a treated group.

2. Methods

Couples attending CFA-Italy gave written consensus for this preparatory treatment. Those enrolled met the following criteria; infertility, as defined as failure to conceive after 12 months and /or previous IVF failures. Patients with previous history of cervical diseases, cervical surgery or infection were excluded. Male gametes were from partners with normospermia, various degrees of oligastenoteratospermia and also sperm extracted from Tese samples. Female partners were stimulated with gonadotrophins to increase the number of oocytes superovulated. Oocyte collection, fertilization, embryo culture and blastocyst vitrification were carried out by standard procedures (Homologous). Other patients received a frozen thawed blastocyst from an oocyte donor using husband spermatozoa (Heterologous). The treated group differed from the control group in the application of high vaginal oxygen and hyaluronic acid. A self cooling Caress Flow (Caress Flow srl, Casette di Fumo, Bologna, Italy) device was used to apply high O₂ (93% ± 3%) at a pressure of 1 ± 0.1 bar at a flow of 2 ± 0.2L/min containing 0.2% of sodium hyaluronate in three 15 minute sessions in the two week period prior to embryo transfer. The first 14 days before embryo transfer, the second 7 days before transfer, the last 24 hrs before transfer. Synchronization and preparation of the uterus followed a standardized protocol for both the control group (n=216) and the Caressflow treated group (n=161).

3. Results

Three hundred and seventy seven patients attending CFA in Naples from September 2024 until April 2025 were used in this study and assigned either to the treated group (n=161) or the control group (n = 216). The average age in the treated group was 38.0 while the average age in the control group was 37.5 years.

Laboratory conditions and blastocyst quality were comparable in the two groups, as were embryo transfer protocols. Serum $\beta$hCG was measured 9 days after embryo transfer with a value of over 90 mlU/mL considered positive.

Overall the data shows a significant increase in the success of blastocyst implantation following treatment with high O₂ and hyaluronic acid (Figure 1), non-significance in the homologous patients (average age of 36.1 for the treated population vs 34.6 for the control group) and high significance in the heterologous patients (average age of 44.4 for the treated patients vs 43.9 for the control population).

4. Discussion

Implantation of the human embryo is a complex dynamic interaction of complimentary tissues (5). Formation of a rapidly expanding blastocyst cavity is a pre-requisite to hatching and depends, in part, on the adequate expression of gap junction, tight junctions and desmosomes (21,22). The trophectoderm cells produce proteolytic enzymes to hatch free of the protective zona pellucida and these may be complimented by proteolytic enzymes produced by the uterus. A molecular dialogue between the hatched blastocyst and the endometrium favours attachment to the epithelium. The blastocyst trophectoderm then secretes hCG which signals the epithelial cells, displacing them, and allowing the embryonic cells to penetrate the basement membrane. Thus, for implantation we may consider three phases, apposition, adhesion and invasion, which must occur at a specific time, i.e. when the uterine epithelium is receptive “the implantation window”.

This window of opportunity is thought to be less than 24 hours long in the human and occurs approximately 6 days after ovulation (5). Attempts to identify this window by biopsy (ERA test) have fallen short (9,23). The blastocyst initially derives nourishment from uterine secretions, but in order to keep growing it must develop its own vascular system, Maternal hormones influence communication between the embryo and the endometrium with cytokines, adhesion molecules, prostaglandins, metalloproteases and angiogenic growth factors all involved.

In this study, we have used as a baseline, frozen thawed blastocysts and, while maintaining patients personalised therapies for individualized endometrial preparation, our prime aim was to determine whether the application of high concentrations of vaginal O₂ and hyaluronic acid could alter the implantation rate in a trial with 377 patients. It is the policy of CFA-Italy to offer PGT-A (evaluation of ploidy) only to patients who request this add-on and we do not routinely screen for chromosomal number in donor embryos. In our study, we have shown that there is a significant increase in implantation rate in the Caress flow treated population with a highly pronounced difference in the heterologous population. Assuming that the blastocysts from the treated group and Caressflow treated group have similar characteristics, the main difference between the heterologous and homologous populations is the age of the receiving patient and probably reflects the inherent differences in uterine physiology of the two patient groups.

Considering the multi-factorial process of early human reproduction it is feasible that the effects of our combined O₂ and hyaluronic acid treatment protocol are also multiple. The reasons for the improved implantation rate in our study at this point can only be the object of conjecture. Briefly, we would like to raise interest in two simple, yet fundamental, pathways. One major factor to consider is the pH of the uterine environment, which appears to be around pH 7.7 in non pregnant women (24). Studies on other mammals, also point to an alkaline uterus with changes correlated with the oestrus cycle in the rat and sow (25,26). A study is in progress at CFA to measure uterine extracellular pH during the oestrous cycle in normo-cycling women. The human oocyte and early cleavage embryo have a pH_i of 7.4 and have poor tolerance to extracellular pH insults below 7.0, however on Day 5 the human blastocyst acquires acid regulating pumps on the plasma membrane and is able to tolerate both acid and alkali conditions [27). Intracellular pH will of course influence all cellular processes. Furthermore, it has been shown that the contractility of the human myometrium is altered by intracellular pH (28) and that the very low vaginal pH of around 4.0 in cycling women is due to the presence of Lactobacillus species. Considering that high concentrations of O₂ and hyaluronic acid are known to improve the vaginal microbiome, increase the hydration of the vaginal mucosa and improve the vascularity of the cervix (16), it would be of interest not only to study the uterine pH but also to classify the uterine microbiome before and after treatment. In fact in a recent study it has been shown that implantation rates may be substantially reduced by the microbial contamination of the embryo transfer catheter with cervico vaginal micororganisms (20).

Although, at present we have no direct evidence that vaginal application of O₂ will influence the uterine cavity, a second, pathway related to high concentrations of O₂ to be considered, is its role in driving redox-signalling and in particular stimulation of expression of growth factors, such as VEGF, reducing inflammatory processes and promoting angiogenesis (29,30).

Finally, since it is known that implantation rates are dependent on the adequacy of the transfer technique (13) we raise the possibility that an improvement in cervical physiology may also account in part for the significant increase in implantation rate.

Conclusion: Our pilot retrospective study on a population of 377 infertile patients treated at CFA, Naples from September 2024 until April 2025 demonstrate that implantation rates of frozen thawed blastocysts may be significantly improved by the vaginal delivery of high concentrations of oxygen and hyaluronic acid in the two week period preceding embryo transfer. The most significant increase in implantation rate was observed in patients receiving donated blastocysts. We discuss the possible reasons for this increase, which may include changing the vaginal and uterine microbiome, altering endometrial extracellular pH, or cervical physiology.