Autologous intrauterine platelet rich plasma instillation in repeated implantation failure in assisted reproductive techniques

INTRODUCTION

The lack of a gestational sac on ultrasonography 5 weeks or more following embryo transfer (ET) with high-quality embryos or following numerous transfers of 10 or more embryos is classified as repeated implantation failure (RIF)¹. Endometrial receptivity, embryo quality, and immunological factors are among the many components implicated in the implantation process². Several techniques to managing RIF have been successful, but there is little agreement on which is the most beneficial. Extra number embryo transfer, assisted hatching, co-culture system, preimplantation genetic screening (PGS), blastocyst transfer, endometrial scratching, sequential transfer, hysteroscopy, tubal disease salpingectomy, endometrial receptivity array (ERA), natural cycle, and immune therapy have all been

used, but there is no proven evidence for these treatments^3,4.

The measurement of endometrial thickness has been used to predict implantation. With increasing endometrial thickness, the likelihood of conception rises. Various investigations have found that the minimal endometrial thickness for embryo transfer is greater than 7 mm. Kasius et al. (2014)⁵ found that ET > 7 mm cases had a significantly higher chance of clinical pregnancy than ET ≤ 7 mm cases (48.1 compared with 23.3%). Conventionally many therapies have been tried in patients with nonoptimal endometrial lining, such as using of Estradiol valerate, intrauterine Granulocyte-colony stimulating factor instillation, use of Sildenafil, Human Chorionic Gonadotropin, low dose aspirin, but they lack consistency in delivering results^6,7,8.

Platelet-rich plasma (PRP) is a fraction of autologous blood plasma with platelet concentrations 4 to 5 times greater than usual⁹. It is prepared from fresh complete blood that has been stored in anticoagulant and processed to separate various components of blood to increase platelets¹⁰. Platelets contain a cocktail of chemical mediators including vascular endothelial growth factor, epidermal growth factor, transforming growth factor, and platelet‑derived growth factor which activating platelets to become bioactive⁹. PRP has been utilized in surgery, wound healing, orthopaedics, and ophthalmology, for over a decade. It induces hard tissues regeneration such as tendons and bones, as well as soft tissues in injectable or gel form, like skin, fat, and mucosa¹⁰. PRP has been indicated to have a role in patients having thin refractory endometrium in limited studies, although its role in patients with implantation failures has received less attention^10,11. PRP is utilized in reproductive medicine to boost implantation rates because it recruits growth factors that favor decidualization and implantation through a paracrine impact. Despite its widespread usage in a variety of medical sectors, PRP's use in reproductive medicine is limited⁹. PRP and its endometrial microvasculature biostimulation effects appear to be favorable to refractory endometrium patients, offering endometrial receptivity increase resulting in an increase in implantation rates, according to new findings. PRP intrauterine injection, according to many writers, can grow thin endometrium and increase pregnancy rates. In women who suffer from thin endometrium, PRP is a relatively recent procedure for increasing the thickness of the endometrium. PRP appears to be safe because it is produced from the sufferer's own blood¹².

In this case, there is a need to evaluate other modalities in this regard, as inadequate endometrial development or vascularity may result in repeated cycle cancellations or recurrent implantation failure, causing not only a psychological but also a financial burden on the patient. This pushes patients into surrogacy, which, given the medicolegal ramifications, may not be a realistic option right now. However, little is known in regard to PRP application's efficacy in the therapy of repeated implantation failure and improvement of pregnancy outcome; therefore, this study aimed to firstly, to assess the role of intrauterine infusion of autologous PRP on ET in ladies who underwent embryo transfer cycles (fresh and frozen) with suboptimal endometrial pattern as assessed by transvaginal ultrasound and secondarily, to study the implantation rate, and clinical outcomes including (rates of chemical pregnancy, clinical pregnancy and miscarriage).

PATIENTS AND METHODS

Criteria

After approval of the ethical committee, the participant patients were fully informed about protocol of the treatment and accordingly they signed consents to participate in the study. The following criteria were used to enroll patients in the research

Inclusion criteria

Age 18-35 years

Women who have had at least three unsuccessful embryo transfer cycles in the past (repeated implantation failure) due to poor endometrial lining.

Exclusion criteria

Age < 18 or > 35 years.

platelets < 150.000/mm³.

Anticoagulant-treated patient.

NSAIDs were taken by the patient for ten days prior to the surgery.

Any major comorbidities or psychiatric illness which might threaten the patient 's consent.

 A cervical or uterine infection that is active,

Women who have a recognized implantation failure reason, like poor embryo quality, Asherman syndrome, or congenital uterine abnormalities

Study procedure

From August 2019 to June 2021, one hundred and four women who had at least three failed embryo transfer cycles (repeated implantation failure) and had high-quality embryos who had been eligible for frozen-thawed and fresh embryo transfer have been eligible to participate in the study. Eight women were excluded for different reasons; 96 were included in the study. Two groups of patients were formed: The study group with 48 patients who underwent intrauterine infusion of PRP (divided into two subgroups frozen-thawed and fresh embryo transfer). While, control group contained 48 patients who did not practiced intrauterine infusion of PRP (divided into two subgroups frozen-thawed and fresh embryo transfer).

A 48 h prior to embryo transfer, a 0.5 ml intrauterine infusion of PRP containing 4-5 times more platelets than a peripheral blood sample is given. Serial transvaginal ultrasound examinations will be done using transvaginal probe of 5 to 9 MHz, on Logic V5 (Logic, GE Healthcare, India) starting from day 7/8, and repeated as required. The maximal distance across the endometrial canal from one basal endometrial interface to the opposite endometrial–myometrial interface after the patient has completely emptied her bladder is used to measure embryo transfer in the uterus's median-longitudinal plane. In women who failed to achieve ET of 7 mm, intrauterine infusion of autologous PRP will be done for the same cycle after receiving written informed consent from the selected women A syringe with an anticoagulant is used to draw a sample of the blood of the patient (10 cm³). PRP is obtained via sequential centrifugation (soft spin 200 g/15 min, then hard spin 600 g/6 min). PRP is infused intrauterine in a volume of 0.5-0.8 ml using an IUI cannula and ultrasound guidance while adhering to all aseptic measures. Repeat ultrasound will be done 72 h later by the same observer and the ET is noted. Second sitting of PRP infusion will be performed in a few patients who fail to show desired results. Embryo transfer is done in the patients who achieve a satisfactory endometrium (ET of 7 mm or more). 2 weeks later, serum beta hCG is measured and appropriate luteal phase support is provided. The rate of positive beta hCG and clinical pregnancy is calculated, as well as the rate of miscarriage. Statistical Package for the Social Sciences (SPSS) version 21 is used to analyze the data obtained. A P value of

Treatment assessment

A skilled ultra-sonographer measures endometrial thickness at the thickest section of the uterus' longitudinal axis. To validate thin endometrium, the thickness was measured three times and the mean of the three measurements had been taken. Endometrial thickness has been evaluated by transvaginal sonography as the primary endpoint, and clinical pregnancy following embryo transfer was the secondary endpoint. When a transvaginal ultrasound revealed an intrauterine gestational sac and an increase in serum β-HCG, clinical pregnancy was verified.

RESULTS

It was a randomized controlled trial in which one hundred and four ladies having a history of failed implantations who have been candidates for frozen-thawed and fresh embryo transfer were eligible to participate in the trial from August 2019 to June 2021. Eight ladies were disqualified for various causes; 96 were able to complete the study and their data were analyzed and included in the study. Two groups of patients were formed: The study group with 48 patients who received intrauterine infusion of PRP (divided into two subgroups frozen-thawed and fresh embryo transfer). While, control group contained 48 patients who did not practiced intrauterine infusion of PRP.

Table (1) presented baseline characteristics summary. The mean age, (BMI), history of past uterine operations and history of previous Failed ET cycles of the study population (control vs. test) were 29.6±0.53, 24.6±0.55, 18(37.5%), 2-5 for control group and 28.4±0.67, 25.6±0.46, 21(43.8%), 2-5 for test group, respectively. The average age, (BMI), history of past uterine operations and history of previous Failed ET Cycles of the study population (control vs. test) were not significantly different.

Mean±S.E; SE, Standard Error; BMI, Body Mass Index; PRP, Platelet Rich plasma; Min.-Max., Minimum-Maximum; * P value ≤ 0.05 is significant; ** P value ≤ 0.01 is significant.

Table 1: Patient’s characteristics, general characteristics and IVF outcomes of patients of the study population.

Table (2) compares the study groups' embryo data as well as their clinical results. The rate of implantation (%), rate of chemical pregnancy (n(%)), rate of clinical pregnancy (n(%)) and rate of misscarriage (n(%)) were 34.8%, 16(33.3%), 11(22.9%) and 5(10.4%) for control group; and 58.5%, 25(52.1%), 22(45.8%) and 3(6.3%) for test group, respectively. The PRP group had a significantly higher rate of implantation and clinical pregnancy outcomes per cycle.

* P value ≤ 0.05 is significant; ** P value ≤ 0.01 is significant.

Table 2: The outcomes of embryo transfer cycles.

Table (3) provided and compared the embryo data as well as the research groups' clinical outcomes (Control and Test “PRP”) as per kind of embryo transfer “fresh ET and frozen ET”. The implantation rate (%), rate of chemical pregnancy (n(%)), rate of clinical pregnancy (n(%)) and rate of misscarriage (n(%)) for fresh ET were 31.8%, 7(29.2%), 5(20.8%), 2(8.3%) for control group; and 55.6%, 10(41.7%), 8(33.3%), 2(8.3%) for test group, respectively. While, for frozen ET were 37.8%, 9(37.5%), 6(25.8%), 3(12.5%) for control group; and 61.2%, 15(62.5%), 14(58.3%), 1(4.2%) for test group, respectively. The rate of implantation , as well as the rates of both chemical and clinical pregnancy, were much greater in the frozen ET “PRP group,” while the misscarriage rate was significantly reduced in comparison to the other group.

Note: Statistical method applied is Chi Square. * P value ≤ 0.05 is significant; ** P value ≤ 0.01 is significant.

Table 3: The outcomes of embryo transfer cycles according to type of embryo transfer.

Table (4) defined the cycle characteristics, including the number of transferred embryos plus embryo transfer stage and thickness of endometrium in the different study groups (Control and Test “PRP”). The No of transferred embryo (mean±s.e), embryo transfer stage including cleavage stage (n(%)) and blastocyst stage (n(%)) were 1.8±0.09, 20(41.7%) and 28(58.3%) for control group; and 2.1±0.11, 22(45.8%) and 26(54.2%) for test group, respectively. The transferred embryos number and embryo transfer stage indicated no significant differences among groups. Moreover, results presented in Table (4), showed that Average Endometrial Thickness (mm) (Mean±S.E) in the case of the first thickness of endometrium in the 13th day of the cycle (D1) the thickness of endometrium following intervention (D2) were 6.5±0.02 and 7.1±0.01 for control group; and 5.8±0.094 and 8.1±0.054 for test group, respectively. The first thickness of endometrium in the 13th day of the cycle (D1) and the thickness of endometrium following intervention (D2) showed no significant differences in control group but in test group was significantly difference.

* P value ≤ 0.05 is significant; ** P value ≤ 0.01 is significant.

Table 4: Cycle characteristics in the transferred cycle.

Results presented in Table (5), showed the impact of Repeated PRP infusion on thickness of endometrium in comparison with First PRP infusion. The Endometrial Thickness (mm) (Mean±S.E) expanded significantly from 5.8±0.09 (Before) to 7.23±0.11 mm (After) with a difference of 1.46±0.03 mm in First PRP infusion group. While in Repeated PRP infusion group endometrial thickness was progressively expanded from 6.7±0.48 (Before) to 8.3±0.60 mm (After) with a difference of 1.62±0.12 mm. All the thickness of endometrium readings in the repeated PRP infusion were significantly different from those in the first PRP infusion (p-value: 0.000).

* P value ≤ 0.05 is significant; ** P value ≤ 0.01 is significant.

Table 5: Endometrial thickness in PRP group.