Application of autologous platelet rich plasma in assisted reproductive technology

MSc Nguyen Huyen Minh Thuy

IVFAS, An Sinh Hospital

What is platelet rich plasma?

Platelet-rich plasma (PRP) is a volume of plasma prepared with more platelets than in normal blood. PRP has been used extensively in medicine. In orthopedic techniques, PRP is used to reduce swelling and promote joint healing in arthritis and tendinitis. The use of PRP in sports injuries provides rapid recovery and complete pain relief. The autologous PRP is derived from the peripheral blood of the patient, which is then put into a centrifuge to remove red blood cells. The concentration of growth agents in PRP is 5-10 times as much as  that of normal blood. When a tissue is damaged, platelets are mobilized to promote healing and attract stem cells to the site of injury. Research results in dentistry, urology and gynecology show that growth agents in PRP are factors that promote the body's natural healing process. In assisted reproduction, PRP is of limited use. This article summarizes current researches and applications of PRP in the field of assisted reproductive technology (ART).

Platelet-rich plasma composition and collection

PRPs extracted from venous blood of patients (about 8-10 ml) are usually supplemented with citric dextrose acid or sodium citrate solution and are put into a centrifuge once or twice depending on the device to collect PRP at an appropriate concentration. Light centrifugation speed and short time aim at separating the whole blood into three layers: the supernatant layer contains plasma, the dark yellow middle layer is platelets, the bottom layer contains red blood cells covered by white blood cells . After the first centrifugation, the speed and time can be increased in the second time to isolate more of the dark yellow layer. Finally, activation factors are added for platelets to release the elements in the cytoplasmic particles.

Cytokines and growth factors are stored in α platelets. These growth factors include: platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), beta conversion growth factor (TGF), fibroblast growth factor (FGF) and interleukin -8. In addition to growth factors, platelets contain other substances, such as fibronectin, vitronectin, and sphingosine-1 phosphate, which are the first factors present in wound healing. PRPs with high concentrations of growth factors are able to accelerate wound healing. Treatment of necrotic tissues, chemotaxis, cell regeneration, proliferation and movement of cells to wounds, synthesis of extracellular matrix, regeneration of wound healing circuits are the goals of growth factors.

Figure 1. Blood components, after centrifugation, (red blood cells, white blood cells and platelets) are separated from the plasma due to their different density (Alves et al. 2018).

The mechanism of PRP helps to improve endometrial quality

For successful embryo implantation, it is necessary to have a combination of good potential embryos and favorable endometrium  to facilitate continuous embryo development. Therefore, besides the culture and selection of optimal embryos, the preparation of a suitable endometrium for the embryo reception process plays an important role in the outcome of the treatment.

In ART, the minimum endometrial thickness required to transfer embryos is 7 mm. A patient with thin endometrium that does not respond to the treatment guideline often leads to cancellation of the embryo transfer cycle. A variety of methods have been developed for the treatment of thin endometrium, including the use of exogenous estrogen, low-dose aspirin, vitamin E, sildenafil citrate, neuromuscular stimulation, granulocyte-colony stimulating factor (G-CSF). However, some women with thin endometrium still do not improve the condition after using these measures. In that case, PRP is a new approach to help improve the thickness of endometrium.

An endometrial tissue contains receptors of growth factors, adhesion molecules, cytokines, growth hormones, PDGF, EGF, lipids and other factors that promote the development of endometrium and embryos. These factors promote the reconstitution of the endometrium tissue, play a role in autocrine and paracrine processes, , and are related to the acceptance of endometrium, nesting and development of the embryo. On that basis, the addition of PRP to the uterine environment can help increase cell proliferation, blood vessels, and regeneration of endometrium, leading to an improvement of endometrium thickness. In addition, PRP may help enhance the expression of genes involved in the nesting process such as progesterone receptor expression gene in the corpus luteum, while reducing IL-1β, IL-6 and IL-8 interleukins expression in endometritis when treated with PRP. Thanks to this, PRP helps improve the ability to receive embryo implants.

Results of PRP application in ART

PRP has been applied to ART to improve the quality of endometrium in patients with thin endometrium or failures in embryo transfer many times even with good embryos. In 2015, Chang et al. reported the first trial using PRP to improve endometrium thickness in patients with thin endometrium. In 2016, D'Alessandro et al. described the standard protocol for PRP application in ART with the following steps: (1) 0.5 ml of PRP injected into the uterus by catheter, (2) endometrium will be tested 48 hours to 72 hours after PRP, (3) If the endometrium is still not suitable, PRP can be pumped 1 to 2 more times.

The results of the first in vitro study assessing the effectiveness of PRP on the proliferation of endometrium cells were reported in October 2016 in ASRM Conference in the USA. In this study, the author demonstrated that PRP helps increase the growth of not only cultured fibroblasts but also mesenchymal cells that are the source of many types of cells, including endometrium ones.

The research by Tandulwadkar et al. (2017) shows that injecting PRP into the uterus promotes angiogenesis. Therefore, when using PRP before transferring embryos to thin MI patients increased the number of blood vessels observed by Doppler ultrasound on regions 3 and 4 of the endometrium. Endometrial thickness and clinical pregnancy rate increased significantly after PRP treatment in this group of patients.

The latest study published by Chang et al. in 2019 on 64 patients with thin endometrium treated with PRP into the uterine cavity showed that PRP improved results of these patients with thin endometrium. Accordingly, the thickness of endometrium was improved compared with the one of the control group (7.65 ± 0.22 mm compared with 6.52 ± 0.31 mm, p <0.05), the rates of implantation and clinical pregnancy were also significantly higher than the those of the control group (27.94% compared to 11.67%; p <0,05 and 44,12% compared to 20%, p <0,05)

In addition to improving results in patients with thin endometrium, many studies have also reported that PRP helps improve ART results in patients who fail to perform multiple nesting even with good quality embryos. A randomized controlled trial of Nazari et al. (2019) found that PRP improved pregnancy outcomes in patients who had multiple implantation failures (44.89% versus 16.66%, P = 0.003). Also in 2019, Kim et al. published an intervention study on a group of patients who failed in embryo implantation after 2 transfers and had thin endometrium. In this study, PRP was pumped 2 to 3 times into the womb of 22 patients. The rate of pregnancy progression and alive births is 20%. The endometrium increased by 0.6 mm on average compared to the period before using PRP. Coksuer et al. (2019) also published a study on 273 patients who failed to implant several times to show that the results of using PRP alone helped improve the endometrium and success rate in ART.

In addition, PRP has the potential to improve ART efficacy in patients with poor ovarian response and early ovarian failure. According to a report by Pantos et al in 2016 in ESHRE conference, the use of PRP in eight women with premenopause and early ovarian failure helped "ovarian rejuvenation" after 1 to 3 months. Afterwards, the patients could receive oocyte and make embryos according to the normal guideline of ART.

In Vietnam, PRP application to improve embryo implantation capacity in patients with thin endometrium and multiple implantation failures is being performed at IVFAS, in conjunction with Mekostem stem cell bank, achieving satisfactory initial results. Currently there are 8 patients with thin endometrium and 8 patients with implantation failure having been treated with PRP and subsequent embryo transfer. As a result, 4 patients in the former group achieve a progressive pregnancy and 6 patients in the latter group are currently pregnant.

Safety of PRP in ART

Using PRP is considered safe because it is derived from the patient's own blood. Currently, there are no reports of side effects when taking PRP on its own. Immune reactions or infectious diseases in blood products are excluded because PRP is derived from autologous blood. At the same time, no cases of infection have been reported after PRP trials. Although PGF has mitotic properties, there is no evidence that growth factors in PRP cause tumors or are related to carcinogen formation. Moreover, the authors have demonstrated that growth factors affect the cell membrane, not the cell nucleus. However, the preparation of PRP requires a lot of processing steps, so there is a possibility of infection. Therefore, all samples must be quality-controlled and sterile in a closed mechanism.

Conclusion

Using PRP is becoming a new approach in assisted reproduction in a non-invasive, simple, effective and safe way. PRP can be used as a means of regenerating IUD tissues in the case of a thin IUD, repeated implantation failure. Using PRP may be a new solution for women with poor ovarian reservation and premature ovarian failure. The risks of PRP associated with bleeding infection and nerve damage are relatively rare. However, well designed studies with large-scaled samples are needed to prove the effectiveness of this method.

References

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