Autologous platelet-rich plasma promotes endometrial growth and improves pregnancy outcome during in vitro fertilization

Abstract

Introduction: This study was to evaluate the effectiveness of PRP in the therapy of infertile women with thin endometrium (≤ 7 mm). Material and methods: Five women undergoing in vitro fertilization (IVF) with poor endometrial response still had thin endometrium (< 7 mm) after standard hormone replacement therapy (HRT) and had to cancel embryo transfer cycle. In addition to HRT, intrauterine infusion of PRP was performed. PRP was prepared from autologous blood by centrifugation, and 0.5-1 ml of PRP was infused into the uterine cavity on the 10^th day of HRT cycle. If endometrial thickness failed to increase 72 h later, PRP infusion was done 1-2 times in each cycle. Embryos were transferred when the endometrium thickness reached > 7 mm. Results: Successful endometrial expansion and pregnancy were observed in all the patients after PRP infusion. Intrauterine PRP infusion represent a new method for the thin endometrium with poor response. Conclusion: This article reported that platelet-rich plasma (PRP) was able to promote the endometrial growth and improve pregnancy outcome of patients with thin endometrium.

Introduction

Successful embryo implantation requires an appropriate embryonic development coincident with a receptive endometrium. In clinical practice, adequate endometrial growth is required for successful implantation. The minimal endometrial thickness required for embryo transfer is 7 mm at the end of follicular phase [1]. Thin endometrium non-responsive to standard treatments is still a challenge in assisted reproductive technique (ART), which usually results in cycle cancellation and unplanned embryo cryopreservation. Various strategies have been developed for the treatment of thin endometrium, including extended use of exogenous estrogen [2], use of low-dose aspirin [3], vitamin E [4] and vaginal sildenafil citrate [5], electroacupuncture [6] and application of granulocyte colony stimulation factor (G-CSF) [7]. However, a number of women with thin endometrium remain non-responsive even these remedies have been performed.

Platelet-rich plasma (PRP) is prepared from fresh whole blood which is collected from a peripheral vein, stored in acid citrate dextrose solution A (ACD-A) anticoagulant and processed to increase platelets by separating various components of blood [8]. Through activating platelets inPRP, cytokines and growth factors (GFs) become bioactive and are secreted within 10 min after clotting. These factors include vascular endothelial growth factor (VEGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) [9]. They can regulate cell migration, attachment, proliferation and differentiation, and promote extracellular matrix accumulation. Nowadays, PRP has been widely applied in different clinical scenarios, such as orthopedics, ophthalmology and wound healing [10] to improve the tissue regeneration. However, little is known regarding the application of PRP in the treatment of thin endometrium. This study was to evaluate the effectiveness of PRP in the therapy of infertile women with thin endometrium (≤ 7 mm).

Patients and methods

Demographics

Five patients with a history of the maximal endometrial thickness of < 7 mm were recruited on the day of administration of human chorionic gonadotropin (hCG), which resulted in fresh embryo transfercancellation and unplanned embryos cryopreservation, and underwent frozen embryo transplantation in the Center for Reproductive Medicine of the Sixth Affiliated Hospital of Sun Yat-sen University from March 2014 to Jun 2014. The age of these patients ranged from 31 years to 39 years. Table 1 summarizestheir characteristics and in vitro fertilization (IVF) cycle outcome.

Table 1.

Patients’ characteristics

Patient	Age (year)	Diagnosis	Endometrium (mm) without/with PRP infusion	Embryotransfer	Pregnancy	Outcome
1a	31	Partial Asherman	5.9/7.0	2 blastocysts	Yes	Ongoing
2b	35	Salpingitis	6.0/7.3	2 blastocysts	Yes (twin)	Ongoing
3c	39	Repeated IVF failures	6.2/7.8	1 blastocyst	Yes	Missed abortion
4d	36	POI	6.6/8.0	2 D3 embryos	Yes	Ongoing
5e	33	Salpingitis	6.4/7.5	2 blastocysts	Yes (twin)	Ongoing

Footnotes: POI = primary ovarian insufficiency, FET = frozen embryo transfer, PRP = platelet-rich plasma, HRT = hormone replacement therapy.

^aThird FET cycle.

Hysteroscopic resection of adhesion treatment was performed when the patient with a history of Asherman syndrome underwent dilation and curettage after miscarriage. Before this cycle, hysteroscopic examination was done again, there were no fibroids, and the endometrial lining was quite little. After the second PRP infusion in HRT cycle, the endometrial thickness was improved, and two blastocysts were transferred with a normally progressing singleton pregnancy.

^bSecond FET cycle.

The endometrial thickness reached to 7.3 mm after single PRP infusion in HRT cycle, and two blastocysts were transferred with a progressing twin pregnancy.

^cFourth ET cycle.

The patient had failed three IVF cycles with seven embryos transferred, and all cycles were ascribed to inadequate endometrial thickness. The endometrial thickness reached 7.8 mm after PRP infusion, and only one blastocyst was transferred. Serum HCG was 226 IU/ml at 3 weeks after embryo transfer, and an intrauterine gestational sac with fetal heart beat was observed by transvaginal ultrasonography in 7^th pregnancy. However, fetal heart was undetectable by ultrasonography at the 9^th week of pregnancy suggesting a missed abortion. The patient had accepted dilation and curettage. Chromosome detection of the trophonema showed 45, XO.

^dSecond FET cycle.

The patient had only 4 antrial follicles, and AMH was 0.46 ng/ml. She was diagnosed as having POI, and just had two cleavage-stage embryos. Fresh ET cycle was cancelled because of inadequate endometrial thickness. In this HRT cycle, the endometrial thickness reached to 7.5 mm with two PRP infusions, and a normally progressing singleton pregnancy was observed.

^eSecond FET cycle, the endometrial thickness increased after PRP infusion in HRT cycle, and two blastocysts were transferred with a progressing twin pregnancy.

In conventional hormone replacement therapy (HRT) treatment cycles, estradiol valerate (Progynova; Bayer Schering Pharma, France) at 6 mg/d was given on day 3 of menstrual cycle.The dosage was steadily increased every 4 days, and the maximal dose was 12 mg/d. All the five patients showed inadequate thin endometrium (5.9-6.6 mm) on the day of administration of progesterone, resulting in cycle cancelation. To evacuate the intrauterine adhesions, hysteroscopic examination was performed. There were no fibroids orpolyps in diagnostic hysteroscopy. Chronically non-responsive thin endometrium resistant to standard treatmentcausedembryo transfer cancellation. They then received administration of PRP, which was approved by the Ethical Review Board of the Sixth Affiliate Hospital of Sun Yat-Sen University, and informed consent was obtained before study.

HRT protocol and PRP preparation

Estradiol valerate (Progynova; Bayer Schering Pharma, France) at 12 mg/d was given on day 3 of menstrual cycle. PRP was prepared from autologous blood by a modified method of Yamaguchi et al [11]. On the 10^th day of HRT cycle, 15 ml of venous blood was drawn from the syringe pre-filled with 5 ml of anticoagulant solution (ACD-A), and centrifuged immediately at 200* g for 10 min. The blood was divided into three layers: red blood cells at the bottom, cellular plasma in the supernatant and a buffy coat layer between them. The plasma layer and buffy coat werecollected to another tube and re-centrifuged at 500* g for 10 min. The resulting pellet of platelets was mixed with 1 ml of supernatant, and then 0.5-1 ml of PRP was obtained. It was infused into the uterus cavity immediately with Tomcat catheter (0.5-1 ml). Endometrial thickness was re-assessed 72 h later. If the endometrial thickness was not satisfied,infusion of PRP was performed 1-2 times. Of five patients, only patient 2 received a single infusion, and the remaining 4 underwent 2 infusions.

Treatment assessment

Endometrial thickness was measured at thethickest part atthe longitudinal axis of the uterus by an experienced ultrasonographer (Figure 1). The thickness was measured thrice to confirm thin endometrium, and the average from 3 measurements was recorded. The primary endpoint was endometrial thickness measured in transvaginal sonography, the second endpoint was clinical pregnancy after embryo transfer. Clinical pregnancy was confirmed when an intrauterine gestational sac was identified by transvaginal ultrasonography and an increase in serum β-HCG was present.

Figure 1.

Ultrasonography in patient 1. A. Quite thin endometrium (only 5 mm) on the day of progesterone administration in traditional treatment. B. Remarkable improvement in endometrial thickness after first infusion ofPRP (7 mm on day 5 before progesterone administration). C. After second infusion of PRP, the endometrial thickness reachednear 8mm on day2 before progesterone administration. D. An intrauterine gestational sac was confirmed by transvaginal ultrasonography at approximately 6 weeks.

Results

The endometrial thickness increased at 48-72 h after PRP infusion in all the patients, and reached > 7 mm on the day of progesterone administration. Patients 1, 2 and 5 underwent transfer of two blastocysts, patient 3 had transferred one blastocyst and patient 4 had transferred two cleavage-stage embryos. Cleavage-stage embryos were transferred on the 3^rd day after progesterone administration, and blastocysts were transferred on the 5^th day after progesterone administration.

All the five patients were pregnant. Patients 2 and 5 had twin pregnancy, and the remaining 3 had one intrauterine gestational sac. Patient 3 experienced a missed abortion at the 9^th week of pregnancy. After induced abortion, detection of chromosomes of the trophonema showed 45, XO. Pregnancies in remaining 4 patients were normally progressing.

Discussion

The major functions of platelets are preventing acute blood loss and repairing vascular walls and adjacent tissues after injury. During wound healing, platelets are activated and aggregate to release granules containing growth factors, such as TGF-β, PDGF, IGF, VEGF, EGF and FGF-2 [12,13], which stimulate the inflammatory cascade and healing process. PRP is defined as aplasma fraction of autologous blood with the concentration of platelets 4-5 times above normal [14]. It is fractionated plasma from autologous blood and contains concentrated platelets. With the functions of platelets, PRP is nowadays widely applied in clinical scenarios, even in mucous tissues such as eye [15] and mouth [16], to improve tissue regeneration. In addition, the combined PRP and mesenchymal stem cells have been widely studied in vitro [17]. This study was to investigate the effectiveness of PRP in improving the endometrial regeneration and the inherent regenerating capacity of endometrium.

In this study, five patients had extraordinarily poor endometrial quality and the endometrium was non-responsive to conventional estrogen therapy, resulting in cycle cancellation, low possibility of pregnancy and heavily emotional distress. After application of PRP, the endometrial thickness was satisfactory in all the patients, who became pregnant after ET. Of them, the pregnancy was normally progressing in 4 women and one had missed abortion.

As the diagnosis of implantation failure is always tentative, clinical efficacy of PRP in this indication is difficult to assess. However, the potential proliferative effect of PRP on the endometrium can be sonographically evaluated. In addition, the endometrial thickness is an important indicator for reproduction [18]. Thus, the endometrial thickness was employed to assess the effect of PRP. The improvement in the endometrial thickness in this study is great likelihood attributable to the infusion of PRP. Although more studies are needed to investigate which degree PRP affects endometrium, and how it improves the reproduction, our findings suggest that endometrial infusion of PRP is able to increase the endometrial thicknessand help the embryo implantation.

Unfortunately, one patient (patient 3) had a missed abortion due to chromosomal abnormality. No evidence attributed to the infusion of PRP, as the other four cases had ongoing pregnancy.

Since PRP is prepared from autologous blood, theoretically there are minimal risks for disease transmission, immunogenic reactions and cancers [19]. Based on the pioneering and long-term clinical experience on the application of PRP in the oralmaxillary field and thousands of patients having received this therapy so far, the use of PRP is considered safe [20]. In this study, no infection or injury was observed.

As we know, our study for the first time reported the application of intrauterine infusion of PRP to improve the endometrial thickness in women with thin endometrium. Increase inendometrial thickness after infusion of PRP in all the patients suggests that intrauterine infusion of PRP represents is a novel strategy for the thin endometrium with poor response to conventional therapy. The findings provide evidence for future randomized, controlled trials with large sample size in this field.

Disclosure of conflict of interest

None.