The use of autoplasma, enriched with platelet-derived soluble factors, when carrying out the programs of in vitro fertilization (IFV) in patients with infertility and chronic endometritis

Infertility in married couples occurs in 15-25% of cases, and it is a serious social problem. According to WHO experts, the frequency of infertility above 15% may affect the demographic indicators of the country. In this regard, this problem attracted the attention of different specialists: endocrinologists, obstetrician-gynecologists, urologists, andrologists, who are engaged in the development of new technologies in treatment. In vitro fertilization (IVF) is one of the most effective methods of infertility treatment. The decrease in IVF effectiveness may be caused by endometrial abnormality (refractory endometrium, hypoplastic endometrium, Asherman’s syndrome) and endometrial insensitivity due to previous chronic subclinical endometritis, repeated uterine curettage, surgical treatment of uterine myoma (conservative myomectomy) and other pathologic processes [1–4].

Recent studies have shown a high prevalence of chronic endometritis (up to 46%) in infertile patients, as well as its association with recurrent abortion and implantation failure [5, 6].

The problem of chronic endometritis treatment remains relevant because of a lack of effectiveness of the existing treatment methods and the indefinite duration of the disease. Despite a wide range of the applied options for conservative treatment of this pathology (antibiotics, synthetic estrogen-progestin drugs), the effect remains relatively low, it limits or reduces the effectiveness of methods for the treatment of infertility, including assisted reproductive technologies.

A new approach in the treatment of this disease is the newly developed methods of regenerative medicine. Currently, the most accessible and promising method is the use of plasma enriched with platelet-derived soluble factors or platelet rich plasma (PRP) [7]. Bio-therapeutic use of autologous peripheral blood has a regenerative effect on damaged tissues due to the increased growth factors, anti-inflammatory cytokines, antimicrobial substances [8]. In the treatment of endometritis, the healing effect is associated with the presence of transforming growth factor β (TGF-β1 and TGF-β2) in PRP, platelet growth factors (PDGF-AA, PDGF-BB, PDGF-AB), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF). These growth factors act synergistically, preventing apoptosis in cells, providing an anti-inflammatory effect and promoting angiogenesis [9]. Because PRP is obtained from autologous blood, the risk of transmission of diseases, immunogenic reactions are minimized. There are studies, which prove the effectiveness of PRP in wound healing and tissue regeneration in orthopedics, dental practice, ophthalmology, reconstructive surgery. The experiments conducted on cows have shown that autologous PRP causes restoration of progesterone receptors, proliferation of endometrial cells, suppression of inflammation caused by bacterial endotoxins [10]. Clinical observations show that PRP has a therapeutic effect on the uterine mucosa in women with Asherman’s syndrome and restores its normal histological structure and function [11]. It is associated with the ability of PRP to stimulate endometrial growth, increase hormone sensitivity and implantation capacity, improve endometrial vascularization, exert anti-inflammatory and antibacterial effect, stimulate endometrial stem cells.

Despite a brief history of development in this area, the use of PRP may be considered as an effective means of restoring endometrial function to ensure the implantation of the embryo during the IVF procedure [12].

Materials and methods

In 2017–2018 we conducted the studies on the possibility of using PRP aimed at increasing the effectiveness of IVF procedures in women with infertility and previous chronic endometritis. The study included 137 patients with infertility associated with chronic endometritis who had indications for IVF. The study was conducted in Healthcare Institution “Clinical Maternity Hospital № 2”, Minsk, Belarus.

The design of the study to be undertaken was reviewed and approved by the ethics committee of Clinical Maternity Hospital № 2, protocol № 9 dated 16.01.2017.

Reproductive-aged patients with infertility and previous diagnosed chronic endometritis undergoing IVF were eligible for inclusion. The patients with poor embryo quality were excluded from the research. All women participating in the study signed informed consent for using PRP in the IVF cycle.

Clinical, instrumental, laboratory and statistical methods were used in the study. The research was a prospective cohort study.

On days 6-9 of the menstrual cycle, aspiration biopsy of the endometrium was performed with the use of “Yunona” aspiration probe or urogenital probe “Paipel”. Morphological study of the endometrium was conducted using two methods: the smear was examined by means of light microscopy and immunohistochemistry. In 87 women (63.5%) participating in the study, the following changes were present in histological responses: lymphoid infiltrates, mainly consisting of lymphoid elements, which were located around the glands and blood vessels; in 74 women (54.9%) there were lymphoid follicles. Stromal fibrosis was diagnosed in 30 women (22.1%), sclerosis of endometrial spiral arteries in 45 women (32.6%). These morphological changes were observed in various combinations, which morphologically confirmed the chronic endometritis in the past medical history. Immunohistochemical studies demonstrated the expression of CD138 (differentiation cluster 138: membrane protein used as an immunological marker of plasma cells).

All patients with chronic endometritis underwent the course of anti-inflammatory therapy according to the current protocols, and after 2-3 months of the treatment, the second endometrial aspiration biopsy was performed with subsequent morphological study.

According to the inclusion and exclusion criteria, two groups of patients were formed for observation. In both groups the ovarian stimulation was performed with the use of long and short protocols of controlled ovarian stimulation, depending on the indications and the concomitant gynecological pathology. Embryo transfer was carried out both in the cycles of ovarian stimulation and in cryocycles.

PRP was not injected during the ovarian stimulation cycle in women in the first group (n = 43). This was a comparison group for the second group of patients. In the second group of patients (n = 94), intrauterine injection of PRP was administered on days 6–10 of the stimulated or synchronized cycle under aseptic conditions, without anesthesia.

The PRP was prepared in the following way: 1) venous blood (9.0 ml) was collected into a test tube containing 3.8% solution of sodium citrate (1.0 ml) as an anticoagulant, and venous blood was centrifuged for 10 minutes at 1500 rpm (286g); 2) under sterile conditions, the plasma obtained after centrifugation was sampled into a sterile tube and re-centrifuged for 10 minutes at 5000 rpm (2800g); 3) the supernatant – platelet-poor plasma (2/3) was removed from the tube, and the remaining platelet-rich plasma (1/3) was collected with the use of sterile intrauterine catheter into a syringe in the volume of 1.0-1.5 ml; 4) 4–5 minutes prior to injection into the uterus, 100 μl of 10% sterile calcium gluconate solution was added to platelet-rich plasma [13].

In this case, the introduction of calcium ions is a platelet-activating factor, resulting in the release of biologically active compounds and growth factors from the vesicle-tubular system of platelets, that is, the enrichment of autoplasma with soluble platelet growth factors. Externally, this was manifested in obtaining a gel-like consistency of autoplasma introduced into the uterine cavity. Application for the invention “Method of preparing patients for in vitro fertilization procedure” is №а20170163 dated 16.05.2017.

Statistical analysis of the obtained data was carried out with the use of parametric and non-parametric techniques, including the methods of descriptive statistics, reliability assessment (Student’s t-test; Chi-square, χ²). The critical value of the significance level was taken to be 5% (p = 0.05). Statistical data processing was carried out using Statistica 10.0 software package.

Results and Discussion

The patients in both groups of study were of the similar age (Chi-square test: χ2 = 2.31, p = 0.128). In group 1 the IVF procedure was performed without the use of PRP (43 patients); and in group 2 it was carried out with the use of PRP (94 patients). Most of the patients were under 35 years old, the proportion of which was 53.5% and 67.0% respectively. Chronic endometritis was morphologically confirmed in patients in both groups. All patients underwent a course of therapy for chronic endometritis according to the current clinical protocols. In 2–3 months after the end of the treatment, aspiration endometrial biopsy was performed as a control test. The signs of chronic endometritis after the treatment were not detected in 16 patients (37.2%) in group 1, and in 35 patients (37.2%) in group 2. Residual morphological signs of endometritis were detected in 27 patients (62.8%) in group 1 and in 59 patients (62.8%) in group 2. In group 1, embryo transfer was performed on day 5 at the blastocyst stage in 27 women (63%), and on day 3 at the cleavage stage in 16 women (37%). In group 2, embryo transfer was performed on day 5 at the blastocyst stage in 54 women (57.4%), and on day 3 at the cleavage stage in 40 women (42.6%). In the first group one embryo was transferred in 16 patients (37.2%), two embryos were transferred in 27 patients (62.7%). In the second group, one embryo was transferred in 52 women (55.3%), two embryos were transferred in 41 women (43.6%) and three embryos were transferred in one (1.1%) woman. Average age of the women, who underwent IFV procedure in the group without the use of plasma was 34.4 years (CI 95%: 32.8–35.9), with the use of plasma — 33.0 years (CI 95%: 32.2—33.9). The analysis of statistical data did not reveal any significant differences in the number of attempts in both groups. In the group without the use of plasma, the average level was 1.6 (1.4-1.8), in the group with the use of plasma - 1.8 (1.5-2.1).

In group 2 with the use of PRP in an IVF cycle, the proportion of women with pregnancies was 64.9% (61 women out of 94): 12.8% with biochemical and 52.1% with clinical pregnancies. In group 1 without the use of PRP in an IVF cycle, the proportion of women with pregnancies was 32.6% (14 patients out of 43): 7% with biochemical and 25.6% with clinical pregnancies (Table 1, 2).

It should be noted that statistically the proportion of women with pregnancy was significantly higher (Chi-square test: χ2yes-no = 12.45, p = 0.0004) in the group of patients with the use of plasma than in the group without the use of plasma (32.6%), and is equal to 64.9%.

It was found that the incidence of deliveries was 41.3 cases (95% CI: 31.2—51.4) per 100 women in the group of patients with the use of PRP, and statistically it was significantly higher (Chi-square test: χ2 = 8.26, p = 0.004; t = 3.28; p <0.05) than in the group of patients without the use of PRP – 16.3 cases per 100 patients (95% CI: 5.2-27.3).

This study shows that the use of PRP affects the inflammatory processes and restoration of endometrial structures. Platelet-derived growth factors, which are released from the platelet vesicular-tubular system when the platelets are activated by calcium ions, have a local anti-inflammatory and regenerative effect on the endometrium damaged by chronic inflammation. Our clinical observations demonstrate, that local infusion of PRP increases the rate of successful embryo implantation in the IVF procedure by 15-25% in comparison with the traditional protocols. In women with infertility associated with chronic endometritis, local infusion of PRP improves endometrial proliferation and receptivity, thereby significantly improving IVF outcomes.

Conclusion

The obtained results contribute to the formation of a new attitude to the introduction of modern methods of regenerative medicine into practice, which modify IVF cycles and, undoubtedly, increase their effectiveness. The simplicity in obtaining the preparation from the patient’s peripheral blood autoplasma, the absence of side effects, the possibility of combining with ovarian stimulation drugs, high efficiency, direct use in the stimulation cycles, and ease of administration may allow this method to be included into IVF protocols in stimulation cycles in women with chronic endometritis in history.