Study Protocol: Association of Platelet-Rich Plasma (PRP) with Endometrium Thickness and Pregnancy Outcome in Women with Recurrent Implantation Failure (RIF).

Infertility is the inability to achieve pregnancy even after 365 days of regular unprotected sexual intercourse. The global infertility rate among reproductive couples is estimated to range between 12.6% and 17.5%. A global estimate shows that infertility affects between 48.5 and 72.4 million couples.[ 1 ] For 20-30% of the infertility cases, only males are responsible and also contribute to 50% of the cases. Many factors are the causes of male infertility, such as age, medications, surgical history, exposure to environmental toxins, and genetic issues.[ 2 ] Fertility in females starts declining around 25-30 years of age. Approximately 1 out of 8 women aged between 15 to 49 undergo infertility treatment. The main causes of female infertility are Premature ovarian insufficiency, polycystic ovary syndrome, endometriosis, uterine fibroids, and endometrial polyps.[ 3 ] Implantation is the process by which a blastocyst sticks to the endometrial wall of the uterus and invades into the epithelium and then the maternal circulation to form the placenta. Before implantation, both the embryo and endometrium have to go through a specific process that is carefully timed and location-specific. The embryo must reach the blastocyst stage while the endometrium prepares itself through hormonal signalling and decidualisation. These changes ensure a supportive environment for implantation, and their precise coordination is essential for the success of early pregnancy.[ 4 ] The endometrium forms the inner lining of the uterus and plays a vital role in the process of reproduction, including regeneration, decidualisation, and support implantation during pregnancy. The thickness of the Endometrium is less than 7 mm on the day of ovulation in a fresh or frozen-thawed embryo transfer (FET) cycle considered a thin endometrium. Thin endometrium results in lower implantation and pregnancy rates, and it results in about 2.4% of the individuals undergoing assisted reproductive technology (ART).[ 5 ] Several techniques have been evaluated for the treatment of infertility including Estradiol hormonal supplementation, low-dose aspirin, vitamin E, vaginal sildenafil, pentoxifylline, tamoxifen, and stem cell therapy. Despite all these techniques, many women face multiple failures due to thin endometrium. Platelet-rich plasma (PRP) emerged as an alternative to increase endometrial thickness and enhance pregnancy outcomes.[ 6 ] Multiple studies have found that PRP is effective in achieving successful implantation in women with thin endometria undergoing FET cycles and fresh IVF-ET cycles.[ 5 ] PRP is obtained by centrifugation of the patient’s blood that has a platelet count above baseline. PRP is injected intrauterine along which numerous proteins, several growth factors (GFs), and cytokines stored in the platelet act on the endometrium through the promotion of cell proliferation and anti-inflammatory properties, which results in successful implantation. PRP treatment is attracting significant interest from researchers because of its distinct benefits. As an autologous biological material, PRP reduces the risk of immune reactions and the transmission of infectious diseases, and its production from peripheral blood makes it less invasive.[ 7 ] So, PRP increases the endometrium thickness along with reducing the chances of recurrent implantation failure in frozen–thawed embryo cycles. In this study, we will discuss how PRP reduces RIF in a patient, which includes women with PCOS and DOR and the preparation of PRP.

Method of recruitment: Self-selection. Recruitment setting: Females facing recurrent implantation failure and have thin endometrium during Frozen embryo transfer and enrolled in Wardha Test Tube Centre, Sawangi, Wardha, Maharashtra and patients will be selected on the basis of the data shown in Table 1 . Inclusion and exclusion criteria The patients will be infused by the PRP, and HRT (Hormonal Replacement Therapy) will be given in the form of Estradiol valerate (6 mg/day) orally from the start of the 2 nd day of the Menstrual Cycle (MC). Then, a Transvaginal ultrasound (TVS) will be done on the 8 th day of the MC. If the EMT (Endometrium Thickness) was less than 7 mm, PRP was prepared. First, their blood plasma will be collected by the doctors by vein puncture in the acid citrate dextrose (ACD) tube (about 10-12 ml). Initially, whole blood will be drawn in the ACD tube that will have an anticoagulant in it (Sodium Citrate). It will be collected on the 13 th day of the MC. Then, it will be poured into the conical tube and kept for the centrifuge at 1200 rpm for 10 min. Do not chill blood at any time or during platelet separation. A first centrifuge will be done to separate the red blood cells, and a second centrifuge will be done to concentrate platelets. After centrifugation, two layers were formed; the upper layer had WBCs and platelets, and the bottom layer had RBCs. Then transfer the supernatant plasma containing platelets into the sterile tube. Then, give a second spin for 10 minutes at 2000 rpm. Then, after centrifugation, it will divide into three layers: Top layer: Platelet-poor plasma. Middle layer: Buffy coat containing concentrated platelets. Lower layer: Containing RBCs. Then buffy coat layers will be carefully separated without disturbing the pallet that contains RBCs. Then, the pallet containing platelets (about 1–2 ml) of PRP will be injected into the intrauterine cavity with the help of a PRP catheter within 1 hr of preparation. Some agents activate the PRP like Calcium Chloride or Thrombin to enhance the release of growth factors. The EMT pattern will be checked every 48 hours, and PRP will be injected if required. When the endometrium reached more than 7 mm, then the cycle continued with the ovulation trigger (using hCG) once the follicle grew to 18 mm in size. The process of oocyte retrieval will be done after 34-36 hours of the trigger. The daily injection of Progesterone 100 mg will be given. After oocyte retrieval, the embryos will be forming and frizzed on day 3 or 5 accordingly. Then embryo will be transferring once your endometrium thickness reaches more then 7 mm. For the patients who will not be given the PRP, their endometrium will be checked through ultrasound, and then oocytes will be retrieved after 36 hours of the hCG trigger. After oocyte retrieval, the embryos will form and frizzed on day 3 or 5 accordingly. Then, embryos will be transferred. After 14 days, the blood sample will be collected, and βhCG will be measured. If the βhCG is more than 25 mIU/ml, then the patient’s pregnancy result will be positive. To standardize PRP treatment protocol in ART. To investigate the role of PRP in addressing recurrent implantation failure due to thin endometrium. To assess the success rate of clinical pregnancy outcome in frozen–thawed embryo transfer after inducing PRP in the patient with RIF. To compare the success rate in clinical pregnancy outcome in patients after infusing PRP and not infusing PRP. To standardize PRP treatment protocol in ART. To investigate the role of PRP in addressing recurrent implantation failure due to thin endometrium. To assess the success rate of clinical pregnancy outcome in frozen–thawed embryo transfer after inducing PRP in the patient with RIF. To compare the success rate in clinical pregnancy outcome in patients after infusing PRP and not infusing PRP. Platelet-rich plasma leads to improved endometrium thickness, reduces recurrent implantation failure in the frozen-thawed embryo transfer cycle, and improves the clinical pregnancy outcome. Standardization of the PRP protocol to get an accurate outcome. Outcome of the clinical pregnancy after the insertion and without insertion of PRP in frozen-thawed embryo transfer. Standardization of the PRP protocol to get an accurate outcome. Outcome of the clinical pregnancy after the insertion and without insertion of PRP in frozen-thawed embryo transfer. Significance Level(α): 0.05 (two-tailed). Power (1 - β):0.80. Effect Size (d): Assume an effect size based on previous. For example, you might expect a difference in pregnancy rates of around 20% between the two groups. Control Group Proportion (p1): Assume 30% for frozen-thawed. Experimental Group Proportion (p2): Assume 50% for fresh. Control Group (Placebo): p 1 = 0.60 Experimental Group (Frozen): p 2 = 0.40 Significance Level(α): 0.05 (two-tailed). Power (1 - β):0.80. Effect Size (d): Assume an effect size based on previous. For example, you might expect a difference in pregnancy rates of around 20% between the two groups. Control Group Proportion (p1): Assume 30% for frozen-thawed. Experimental Group Proportion (p2): Assume 50% for fresh. Control Group (Placebo): p 1 = 0.60 Experimental Group (Frozen): p 2 = 0.40 Where: Z α/2 = 1.96 Z β = 0.84 Z α/2 = 1.96 Z β = 0.84 Substituting the new values: Z α/2 + Z β = 2.80 (Z α/2 + Z β ) 2 = 7.84 p1(1 - p1) = 0.60 × 0.40 = 0.24 p2(1 - p2) = 0.40 × 0.60 = 0.24 p1(1 - p1) + p2(1 - p2) = 0.24 + 0.24 = 0.48 (p2 - p1) 2 = (0.40 - 0.60) 2 = 0.04 Z α/2 + Z β = 2.80 (Z α/2 + Z β ) 2 = 7.84 p1(1 - p1) = 0.60 × 0.40 = 0.24 p2(1 - p2) = 0.40 × 0.60 = 0.24 p1(1 - p1) + p2(1 - p2) = 0.24 + 0.24 = 0.48 (p2 - p1) 2 = (0.40 - 0.60) 2 = 0.04 Thus: This suggests you would need approximately 95 patients per group for adequate power. Total sample size = 95 + 95 = 190 patients. Individual patient will be selecting through clinical report and patient permission. The patient with recurrent implantation failure and thin endometrium measured through ultrasound undergoing IVF treatment will includes and patients in study group will infuse with the PRP and patient in control group will be giving placebo.

The study will follow a structured process that starts with the enrolment of the participant, then assignment, obtaining consent, then allocation, intervention, follow-up, analysis, and then the First, the participants will be screened and then those who meet the criteria will enroll in the study. The eligible participants will be divided into two different groups, I.e, the study group and the control group,by a non-randomized method. Participate in the study group will exposes to intra uterine PRP and the control group will expose to placebo. During the follow up process, the data will be collecting by monitoring the endometrial thickness, implantation and pregnancy rate in both the groups. Statistical analysis will be conducting on the participates who will complete the study by comparing theclinical pregnancy rate in each groups. Follow-up will up to the evaluation of clinical pregnancy. The data analysis will incorporate absolute numbers and statistical estimation method which will includes confidence intervals to determine the precision of the finding. Overall, the study will demonstrate that PRP improves the endometrium thickness, reduces the recurrent implantation failure and improves the clinical pregnancy rate and also increases the pregnancy rate in the patients who will be infusing with the PRP. It will be highlighting that PRP is a safe, cost-effective andenhances endometrial receptivity.

According to the study conducted by Yunying Lin et al . in 2021, The study shows that the endometrium plays a significant role in increasing endometrium thickness and embryo implantation. Patients with thin or dysfunctional endometria face challenges in achieving pregnancy. PRP has some growth factors, such as PDGF, which help in the proliferation of the endometrium. PRP helps to regulate the inflammatory process, which improves embryo implantation. In some studies, it is shown that PRP is injected before embryo transfer. PRP is beneficial in patients with recurrent implantation failure and thin endometrium. In this study, they didn’t mention when to take the PRP or in what dosage. They have studied on a smaller sample size and lack of a control group. Overall, this study shows that the PRP improves the endometrium thickness and improves the implantation rate.[ 8 ] According to the study conducted by Maryam Eftekhar et al . in 2018, they studied 83 women with thin endometria for the FET cycle. The study was conducted in two groups: the PRP group (n = 43) and the control group (n = 40). Out of them, 10 women in the control group and 7 in the PRP group had no embryo transfer due to very thin endometrium. In the PRP group, 23 women only needed 1 infusion of PRP, and 10 needed 2 infusions of PRP. The study concluded that the endometrium increased from 6.12 mm to 8.80 mm in the conceived PRP group, from 6.06 mm to 8.54 mm in non-conceived women, from 6.10 mm to 8.06 mm in the conceived control group and from 6.05 mm to 8.04 mm in the non-conceived group. The implantation rate in PRP is 21%, and in the control group, it is 9.37%. The cycle cancellation rate was lower in the PRP group. PRP was injected in the uterine cavity on the 10 th day of the FET cycle, and if the Endometrial thickness (EMT) failed to increase even after 72 hours, PRP injections were given 1-2 times in each cycle. If the endometrium thickness reaches more than 7 mm, embryos are transferred. So, the study suggests that the PRP can successfully increase the endometrium thickness and improve the pregnancy outcome in women with thin endometria. It is also a safe procedure with minimal risks, making it more effective for fertility treatment.[ 9 ] It will improve the endometrium thickness and increase the pregnancy rate in the RIF and thin endometrium patient who will infuses with the PRP. PRP will be very cost effective as it is affordable and low risk adjunct to improve endometrium receptivity. It will improve the endometrium thickness and increase the pregnancy rate in the RIF and thin endometrium patient who will infuses with the PRP. PRP will be very cost effective as it is affordable and low risk adjunct to improve endometrium receptivity. There are no conflicts of interest.