Intrauterine autologous platelet-rich plasma treatment in women with at least two implantation failures: A retrospective cohort study

Abstract Background Finding the most effective way to improve implantation rate in women who are receiving assisted reproductive technology treatment is still a challenge. Objective This study aimed to assess the pregnancy outcomes of intrauterine platelet-rich plasma (PRP) therapy in women with a history of at least 2 implantation failures. Materials and Methods In this retrospective cohort study, data of 852 women who were candidates for frozen-thawed embryo transfer was extracted from their medical records from April 2017 to September 2021 at Yazd Reproductive Sciences Institute, Yazd, Iran. Of these, 432 received intrauterine PRP treatment 48 hr before transfer (PRP group), and the results of the pregnancy outcomes compared with 420 of the control group who did not receive the treatment before transfer. Results Pregnancy outcomes, including chemical, clinical, ongoing pregnancy, and live birth rate were statistically significant in the PRP group (p < 0.001). However, when categorized according to the implantation history, this significant improvement in all 4 was only seen in women with at least 2 prior implantation failures. In women with a history of only one implantation failure, PRP therapy significantly improved the ongoing pregnancy and live birth rate (19.5%, p = 0.04). Also, in women who received donor eggs and had repeated implantation failure, PRP improved pregnancy outcomes clinically but not statistically (p = 0.15). Conclusion PRP seems to be effective in improving the pregnancy rate in women with a history of 2 or more implantation failures and also shows an increase in the live birth rate in women with only one implantation failure.


Introduction
Implantation is a sophisticated matter that needs both a good quality embryo and a receptive and prepared endometrium to work in harmony.
A successful implantation is considered a visualization of the pregnancy sac at the 5 th wk.On ultrasound after transferring the embryo, the factors that are involved in the process can be categorized into 4 main groups: embryonic quality; embryo-endometrial cross-talk; the regulation of maternal immunologic mediators; and endometrial receptivity.If any of these face difficulties despite the presence of a good-quality embryo, implantation failure might occur (4)(5)(6).(11).
Intrauterine infusion of PRP as a rich source of several cytokines and growth factors, including insulin-like growth factor I, II, fibroblast growth factor, and interleukin 8, just to name a few, may affect the process of growth in the endometrium and its receptiveness to the embryo (12)(13)(14)(15).
This study aimed to assess the effectiveness of intrauterine infusion of PRP on pregnancy outcomes in women with a history of at least 2 implantation failures.

Materials and Methods
In this retrospective cohort study, data from 852 frozen-thawed embryo transfer (FET) candidates aged between 18 and 42 yr was extracted from their medical records from April 2017 to September 2021 in Yazd Reproductive Sciences Institute, Yazd, Iran.
Women with known uterine anomalies (congenital or acquired), thrombophilia, and uncontrolled endocrine disorders such as hypothyroidism and hyperprolactinemia were excluded.
To prepare the endometrium for transfer, both the control (n = 420) and PRP group (n = 432) received 6 mg/day of estradiol valerate from the second day of menstruation and on the 13 th day of menstrual cycle vaginal sonography was performed.If the endometrial thickness was at Intrauterine autologous PRP in implantation failure least ≥ 7 mm then 400 mg vaginal progesterone was prescribed every 12 hr for 3 days.
In the PRP group 2 days before transfer, 8.5 ml of blood was taken into an anticoagulated syringe with anticoagulant citrate dextrose-A solution and then centrifuged at 1600 g for 10 min.The plasma layer and buffy coat were transferred into another syringe and centrifuged again at 3500 g for 5 min.The final product would then be 1.5 ml PRP with 4-5 times platelet concentration more than peripheral blood.Then 0.5-1 cc of PRP was administered intrauterine (14).
All women returned for embryo transfer after 3 days of progesterone administration.Estradiol tablets and vaginal progesterone were continued until the 10 th wk of pregnancy.
On the 14 th day of embryo transfer, the level of beta human chorionic gonadotropin hormone was measured, and any level above 50 IU/L was considered a positive chemical pregnancy.
Detection of fetal heart activity 2 wk after a positive beta-human chorionic gonadotropin hormone confirmed a clinical pregnancy.Ongoing pregnancy was defined as an established pregnancy after the 12 th wk of gestation.The live birth rate was considered the birth of a live fetus after 28 gestational weeks.
Factors such as age, type of ART cycle, number, and quality of retrieved oocytes and embryos were analyzed.

Sample size
A total of 432 women received PRP treatment 48 hr before transfer.To ensure a suitable power for the study, we allocated a 1:1 ratio for the control group.A total of 3351 cases of FETs were matched in our inclusion and exclusion criteria in this period.From this, we chose the first of every 8 files, using systematic sampling and 420 women files enrolled in the control group.
From 432 women in PRP group, 217 cases had a history of at least 2 prior implantation failures, and from 420 women in control group 101 had a history of 2 or more implantation failures.

Ethical considerations
The study protocol was approved by the Ethics Committee of Yazd Research and Clinical Center for Infertility, Yazd, Iran (Code: IR.SSU.RSI.REC.1401.006).

Results
In this study, data from 852 women were analyzed, with 432 in the PRP group and 420 in the control group.Of these, 318 had a history of 2 or more implantation failures, with 217 in the PRP group and 101 in the control group.Demographic characteristics between the PRP and control groups showed no significant differences, irrespective of implantation failure history (Tables I and II However, when considering the history of implantation failure, improvements in all 4 categories were evident only in women with at least 2 prior failures.For those with a history of one implantation failure, PRP significantly improved ongoing pregnancy and live birth rates, but chemical and clinical pregnancy rates lacked statistical significance (Table IV).
The study also assessed outcomes for 269 women who received donor eggs, with 165 undergoing PRP treatment before transfer.
Chemical and clinical pregnancy rates showed statistical improvement in the PRP group.
Although ongoing pregnancy and live birth rates were higher, the results were not statistically significant (Table V).

Discussion
In this study, we assessed the pregnancy outcomes in 432 women who underwent intrauterine PRP treatment 48 hr before frozen-thawed embryo and compared it to a control group of 420.We found out that overall chemical, clinical, ongoing pregnancy, and live birth rates were higher in the PRP group, and the results were statistically significant.
In a more detailed analysis, it is determined that these parameters were only statistically significant if there was a history of at least 2 implantation failures present.Also, it was an interesting observation that although there was not a significantly higher pregnancy rate in women with a history of only one implantation failure, PRP treatment improved the likelihood of the live birth rate in this group.
In a 2023 randomized clinical trial, we compared the effect of PRP therapy on 33 women with a history of at least 2 prior implantation failures with the control group.We found that the PRP treatment before embryo transfer improved ART outcomes, that is, chemical, clinical, and ongoing pregnancy rates, but it was not statistically significant.It is worth mentioning that the size of our population study was small, and we did not evaluate the effect of the PRP treatment on the live birth rate and abortion rate (14) (20).On the other hand, a 2023 meta-analysis, recorded that there is little confidence that administration of the PRP had any significant effect on chemical and clinical pregnancy or live birth rates (21).This is in contrast to the Abd Elsalam Elgendy meta-analysis in 2023, which has reported improved chemical and clinical pregnancy rates as well as endometrial thickness in women with RIF after the PRP administration.It's worth mentioning that they did not assess its effect on the live birth rate (22).
According to all that mentioned above, it seems that whether the PRP could be a good approach to improve pregnancy outcomes in women with or without RIF needs more investigation and time to conclude.
Our results showed that in women who received donor eggs and had a history of repeated implantation failure, the PRP treatment also improves pregnancy outcomes as well.However, for live birth and ongoing pregnancy rates, it was not statistically significant which seems to be due to the small number of the control group (only 26).We could not find any study that would address this specific matter (intrauterine PRP treatment in women with RIF who used donor eggs).Considering that we can almost exclude the ovarian factor of infertility in this group, conducting more studies with sufficient sample sizes to establish results could be quite helpful in providing information about the effectiveness of the PRP in improving the chance of implantation.

Conclusion
Based on this study's results, it seems that PRP could be a beneficial approach in women with RIF and might be considered in patients with only one implantation failure to improve the live birth rate.
Several methods have been considered for improving the implantation rate in assisted reproductive technology (ART), but the most effective one remains unclear.Intrauterine infusion of peripheral blood mononuclear cells, growth hormone, and granulosa colonystimulating factor are just a few examples.Tubal ligation and salpingectomy for the treatment of hydrosalpinx, assessing the uterine cavity with hysteroscopy, and performing endometrial scratching are other options.In the lab environment, transferring the embryo in the blastocyst stage and performing preimplantation genetic screening on embryos before the transfer also have been considered (7-10).It has been shown that endometrial mesenchymal stem cells and endometrial stromal fibroblasts as well as other cellular components have a key role in blastocyst implantation and promote cell adhesion and immunological responses, it seems PRP enhances their movement during the process and therefore improve the chance of pregnancy.On the other hand, PRP also has stimulatory effects on the function and production of various growth factors that are involved in cell attraction, migration, and transformation as well as vascularization and inflammation, which all are crucial steps in a successful implantation Data were analyzed using the SPSS software (Statistical Package for Social Sciences, SPSS Inc version 2.0., Chicago, Illinois, USA).The Chi-square, Mann-Whitney, and student's t tests were used to evaluate the relation between variables.P-value < 0.05 was considered statistically significant.

Table III .
Comparison of pregnancy outcomes between the 2 study groups

Table IV .
Comparison of pregnancy outcomes between the 2 study groups considering implantation failure history

Table V .
Comparison of pregnancy outcomes between the 2 study groups considering donor egg recipients .
Deng et al. reported results in the same lane in their 2022 meta-analysis