Sunita Chandra, M.D., Suhani C, M.S. Et Al. 2019
To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Abstract BACKGROUND: During assisted reproductive technology (ART) treatments, some cycles are cancelled due to inadequate endometrial growth as Endometrium is one of the main factors in pregnancy . Despite the advancements in assisted reproductive technologies, repeated implantation failure (RIF) still remains a challenging problem for patients and clinicians. The aim of the present study was evaluate the impact of intrauterine infusion of autologous platelet-rich plasma (PRP) on pregnancy outcome in patients with repeated implantation failure.
METHODS: 119 patients with history of inadequate endometrial growth in frozen-thawed embryo transfer (FET) cycles were recruited into the study. Reasons for non-participations of 11 patients were tabled. Intrauterine infusion of PRP was performed. Endometrial thickness was assessed. Chemical and clinical pregnancies were reported.
RESULTS: In all patients, endometrial thickness increased after PRP and embryo transfer was done in all of them. 49 patients were pregnant. According to this study, it seems that PRP was effective for endometrial growth in patient with thin endometrium.
CONCLUSIONS: Our study demonstrates that PRP was effective for endometrial growth in patients with thin endometrium and has the potential to improve chemical and clinical pregnancy rate of the infertile women with thin endometrium in frozen-thawed embryo transfer cycle.
platelet-rich plasma, thin endometrium, frozen-thawed, embryo transfer, pregnancy rate
Thin endometrium is relatively frequent in women with previous trauma of the uterus (cesarean sections, repetitive curettage), patients subjected to antitumoral treatments in childhood (Radiotherapy, Chemotherapy, Surgery), women affected by Asherman's syndrome, chronic infections (endometritis, Pelvic Inflammatory Disease) and inadequate blood flow (stress, malposition of uterus, fibrosis), patients with low estradiol values or excessive use of Clomiphene Citrate.
Chronically thin endometrium resistant to standard treatments affects a small number of patients undergoing IVF. This problem, nevertheless, is of considerable importance because endometrium below 7 mm in thickness is widely considered sub-optimal for transfer and associated with reduced pregnancy chances 1 (Casper, 2011; Singh et al., 2011; Revel, 2012). Various remedies have been proposed, including extended estrogen administration if time allows 2 (Chen et al., 2006), low-dose aspirin 3 (Weckstein et al., 1997) and treatment with pentoxifylline and tocopherol 4 (Le ́de ́e-Bataille et al., 2002) and with vaginal sildenafil citrate 5 (Sher and Fisch, 2002). However, even utilizing these remedies, a small number of women remain unresponsive.
Buy BabyChakra Products: http://babyc.in/l/XP5p Get 10% discount use code:10COM
We often see patients with a thin endometrium in ART cycles, in spite of standard and adjuvant treatments. Improving endometrial growth in patients with a thin endometrium is very difficult. Without adequate endometrial thickness these patients, likely, would not have reached embryo transfer.
Endometrium is one of the main factors in implantation and pregnancy. Pregnancy rate is increased with growing endometrial thickness. In several studies, the minimum endometrium thickness for embryo transfer was reported to be 7 mm 6 (El-Toukhy et al., 2008; Richter et al., 2007). Several methods are performed for endometrial preparation in frozen-thawed embryo transfer (FET) cycles, and there is little consensus on the most effective route. Some FET cycles are cancelled due to thin endometrium despite routine treatment, and there is no established protocol for this condition. Extended estrogen treatment and adjuvant therapy, such as low dose Aspirin, vaginal Sildenafil, Pentoxifylline and intrauterine perfusion with granulocyte-colony stimulating factor (G-CSF) have been used for thin endometrium, but there isn’t any proved evidence in this treatment 7 (Barad et al., 2014; Chang et al., 2015; 8 Eftekhar et al., 2014; Gleicher et al., 2013; Groenewoud et al., 2013; 9 Lebovitz & Orvieto, 2014; Xu et al., 2015).
PRP is a new approach that has been suggested for the treatment of thin endometrium 10 (Chang et al., 2015). PRP is blood plasma prepared from fresh whole blood that has been enriched with platelets. It is collected from peripheral veins and contains several growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF ), transforming growth factor (TGF) and other cytokines that stimulate proliferation and growth. Recently, PRP has been used in several medical conditions in ophthalmology, orthopedics, surgery and wound healing but it’s efficacy in endometrial growth has not been fully elucidated. The aim of this study was to evaluate the effectiveness of intrauterine infusion of PRP in the treatment of thin endometrium in FET cycles 11 (El- Anwar et al., 2016; Lee et al., 2016; Maria-Angeliki et al., 2015; Picard et al., 2015; Ronci et al., 2015; 12 Rossi et al., 2016; Sadabad et al., 2016).
MATERIALS AND METHODS This interventional, non-randomized clinical trial was performed at the Rajendra Nagar Hospital & IVF Centre, Lucknow, India between August 2018 and July 2019. The study was approved by the Ethics Committee of the Hospital. Informed written consent was obtained from all couples. 119 women with endometrial thickness below 7mm at 12PthP-13PthP day of frozen-thawed embryo transfer cycle were allocated in this study. Patients less than 25 years, older than 45 years, those with BMI>30 Kg/mP2P, history of endocrine disorders, systemic diseases, Bleeding diathesis, Previous uterine surgery (miomectomy, cesarean section, etc...),Platelet count < 105/Μl , Hemoglobin < 10 g/dL , severe endometriosis, repeated (≥3) implantation failure, repeated (≥3) abortion, congenital or acquired uterine anomaly, were excluded from the study.
In 108 patients( Reasons for non-participations of 11 patients were tabled ), Hysteroscopic examination was performed before the cycle, if it had not been done previously. Hormone replacement therapy (HRT) was performed for endometrial preparation in all participant: estradiol valerate 6 mg/d was started on the 2nd or 3rd day of the mensural cycle and it was increased to 8 mg/d on day 9-10 because of inadequate endometrial growth (< 7 mm). PRP was performed on day 11-12 in all the patients due to thin
endometrium and it was repeated on day 13-14. During the cycle, whenever the endometrial thickness was more than 7 mm, suppository progesterone 400 mg twice-a-day was started and embryo transfer (ET) was carried out per embryonic stage. Estradiol valerate and progesterone supplementation were continued for 2 weeks after ET and if the serum βHCG was positive, hormone supplementations were continued until 12 weeks of gestation. Transvaginal ultrasound was performed by one machine. Endometrial thickness was measured at the thickest part in the longitudinal axis of the uterus.
PRP was obtained from a fresh whole blood collected from a peripheral vein; the blood sample was centrifuged at 1500g (RCF) for 10 minutes and the repeated reversal of the tube will allow obtaining the PRP at the concentration required. Then 0,5-1ml of PRP was infused into the uterine cavity through a Tomcat catheter. The endometrial thickening was evaluated by ultrasonography 24-48h after the instillation and, if the endometrial lining reaches 7mm the Embryo-transfer was to be arranged.
Primary Outcome Measures :
1. Endometrial thickness [ Time Frame: 24-48h after the intrauterine PRP infusion ] 2. Endometrial thickness > 7 mm measured by means of transvaginal ultrasound
Secondary Outcome Measures :
1. Positive pregnancy test rate [ Time Frame: Approximately 3 weeks after treatment ]
Positive pregnancy test rate after Embryo-transfer 2. Implantation rate [ Time Frame: Approximately 6 weeks after treatment ]
defined by number of gestational sacs seen on early pregnancy 6-week ultrasound divided by number of embryos transferred
Other Outcome Measures:
1. Clinical pregnancy rate [ Time Frame: Approximately 8 weeks after treatment ]
Defined by the number of fetal poles with heartbeat seen on 6-week ultrasound divided by the number of embryos transferred 2. Return to spontaneous period [ Time Frame: Approximately 1 to 3 months after treatment ]
Records of a menstrual flow diary (Menstrual Assessment Chart) for 1-3 months after treatment
The main outcome was endometrial expansion and the secondary outcomes were chemical and clinical pregnancies, determined by positive serum βHCG, 2 weeks after ET and the presence of fetal hear beat in the transvaginal ultrasound 5 weeks after ET.
A total of 108 patients with a history of FET cancellation due to thin endometrium were recruited into the study. Uterine cavity abnormalities were not detected before starting the cycle. All the participants needed PRP in the treatment cycles due to inadequate endometrium growth. Endometrial thickness increased at 48 h after the first PRP and reached more than 7 mm after the second PRP in all patients. Embryo transfer was then carried out for all of them. 49 patients were pregnant and in 46 of them the pregnancy progressed normally.
DISCUSSION The preparation of PRP is an outpatient procedure that involves a blood draw, preparation of the PRP, and the injection of PRP into the diseased area. Multiple methods have been developed for PRP preparation, with variation in the speed and timing of centrifugation. The following steps present a representative method of preparing PRP: (1) venous blood (15–50 mL) is drawn from the patient’s arm in anticoagulant- containing tubes; (2) the recommended temperature during processing is 21 ̊C–24 ̊C to prevent platelet activation during centrifugation of the blood; (3) the blood is centrifuged at 1,200 rpm for 12 minutes; (4)
the blood separates into three layers: an upper layer that contains platelets and white blood cells, an intermediate thin layer (the buffy coat) that is rich in white blood cells, and a bottom layer that contains red blood cells; (5) the upper and intermediate buffy layers are transferred to an empty sterile tube. The plasma is centrifuged again at 3,300 rpm for 7 minutes to help with the formation of soft pellets (erythrocytes and platelets) at the bottom of the tube; (6) the upper two-thirds of the plasma is discarded because it is platelet-poor plasma; (7) pellets are homogenized in the lower third (5 mL) of the plasma to create the PRP; (8) the PRP is now ready for injection. Approximately 30 mL of venous blood yields 3–5 mL of PRP; (9) the affected area is disinfected before the PRP injection; (10) providing assurance to the patient and discussing the procedure make the injection easier and less painful; (11) PRP stimulates a series of biological responses, and the injection site may become swollen and painful for roughly 3 days.
PRP is autologous blood plasma that has been enriched with platelets at about 4-5 times more than the circulating blood. PRP can stimulate proliferation and regeneration with a large amount of growth factors and cytokines, including PDGF, TGF, VGEF, EGF, fibroblast growth factor (FGF), insulin-like growth factor I, II (IGF I, II), interleukin 8 (IL- 8) and connective tissue growth factor (CTGF). Currently, PRP infusion is being increasingly used in several fields in medicine such as nerve injury, osteoarthritis, chronic tendinitis, bone repair and regeneration, cardiac muscles, alopecia, plastic surgery and oral surgery, but there is limited experience in gynecology and obstetrics 13 (Alcaraz et al., 2015; Borrione et al., 2010; Patel et al., 2016; Yu et al., 2011).
Adequate endometrial thickness is a main factor for implantation and pregnancy. Women with persistent thin endometrium often do not undergo embryo transfer. Several methods have been described for endometrial preparation but there is not any definitive method yet. In recent years, intrauterine infusion of G-CSF has been studied but inconsistent results have been reported. Some researchers reported that G-CSF favors endometrial growth and pregnancy. G-CSF is a cytokine that stimulates neutrophilic granulocyte differentiation and proliferation, it may induce endometrium proliferation and growth, thus improve pregnancy outcome. According to this hypothesis, local infusion of PRP that contains several growth factors and cytokines may improve endometrial growth and receptivity. PRP is collected from autologous blood sample, so in comparison to G-CSF, PRP is more accessible and affordable 14 (Gleicher et al., 2011; Lucena & Moreno-Ortiz, 2013). This study revealed the efficacy of PRP on endometrial growth. Adequate endometrial growth was found in all the participants after two PRP infusions in all patients who had a history of cycle cancellation due to thin endometrium. At the present, there is limited evidence in this regard. PRP is a safe procedure, with minimal risks of transmission of infectious disease and immunological reactions since it is made from autologous blood samples.
CONCLUSION Our study demonstrates that PRP can expand endometrial thickness with recruiting growth factor to the endometrium and has the potential to improve chemical and clinical pregnancy rate of the infertile women with thin endometrium in frozen-thawed embryo transfer cycle, PRP is an innovative therapeutic modality, as it is affordable, simple, easily performed, and effective. It is also a noninvasive modality with promising results and no side effects.PRP is a safe procedure, with minimal risks of infectious disease transmission and immunological reactions since it is made from autologous blood samples. We suggest that Large randomized controlled studies are required to confirm its efficacy and safety in various gynaecological disorders.
The authors would like to thank all the women for their participation in this study. We would also like to thank the personnel of the Rajendra Nagar Hospital & IVF Centre.
This study was funded by the Revolving Fund of the Rajendra Nagar Hospital & IVF Centre, Lucknow, India
CONFLICT OF INTEREST
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
1. Casper RF. It’s time to pay attention to the endometrium. Fertil Steril 2011; 96:519 – 521. 2. Chen MJ, Yang JH, Peng FH, Chen Su, Ho HN, Yang YS. Extended estrogen administration for women with thin
endometrium in frozen-thawed in-vitro fertilization. J Assist Reprod Genet 2006; 23:337 – 342. 3. Weckstein LN, Jacobson A, Galen D, Hampton K, Hammel J. Low-dose aspirin for oocyte donation recipients with a
thin endometrium: prospective randomized study. Fertil Steril 1997;68:927 – 930. 4. Le ́de ́e-Bataille N, Olivennes F, Lefaix JL, Chaouat G, Frydman R, Delanian S. Combined treatment by pentoxifilline and tocopherol for recipient women with a thin endometrium enrolled in an oocyte donation program. Hum Reprod 2002;17:1249 – 1253 5. Sher G, Fisch JD. Effect of vaginal sildenafil on the outcome of in vitro fertilization (IVF) after multiple failures
attributed to poor endometrial development. Fertil Steril 2002;78:1073 – 1076. 6. El-Toukhy T, Coomarasamy A, Khairy M, Sunkara K, Seed P, Khalaf Y, Braude P. The relationship between
endometrial thickness and outcome of medicated frozen embryo replacement cycles. Fertil Steril. 2008;89:832-9. PMID: 17681313 DOI: http://dx.doi.org/10.1016/j.fertnstert.2007.04.031 7. Barad DH, Yu Y, Kushnir VA, Shohat-Tal A, Lazzaroni E, Lee HJ, Gleicher N. A randomized clinical trial of en dometrial perfusion with granulocyte colony-stimulating factor in in vitro fertilization cycles: impact on endometrial thickness and clinical pregnancy rates. Fertil Steril. 2014;101:710-5. PMID: 24424357 DOI: http://dx.doi.org/10.1016/j.fertnstert.2013.12.016 8. Eftekhar M, Sayadi M, Arabjahvani F. Transvaginal perfu sion of G-CSF for infertile women with thin endometrium in
frozen ET program: A non-randomized clinical trial. Iran J Reprod Med. 2014;12:661-6. PMID: 25469123 9. Lebovitz O, Orvieto R. Treating patients with “thin” endometrium - an ongoing challenge. Gynecol En docrinol.
2014;30:409-14. PMID: 24693854 DOI: http://dx.doi.org/10.3109/09513590.2014.906571 10. Chang Y, Li J, Chen Y, Wei L, Yang X, Shi Y, Liang X. Autologous platelet-rich plasma promotes endometrial growth
and improves pregnancy outcome during in vitro fertilization. Int J Clin Exp Med. 2015;8:1286-90. PMID: 25785127 11. El-Anwar MW, Nofal AA, Khalifa M, Quriba AS. Use of autol ogous platelet-rich plasma in complete cleft palate repair.
Laryngoscope. 2016;126:1524-8. PMID: 27075516 DOI: http://dx.doi.org/10.1002/lary.25868 12. Rossi LA, Molina Rómoli AR, Bertona Altieri BA, Burgos Flor JA, Scordo WE, Elizondo CM. Does platelet-rich plasma decrease time to return to sports in acute muscle tear? A ran domized controlled trial. Knee Surg Sports Traumatol Ar- throsc. 2016. [Epub ahead of print] PMID: 27085364 DOI: http://dx.doi.org/10.1007/s00167-016-4129-7 13. Alcaraz J, Oliver A, Sánchez JM. Platelet-Rich Plasma in a Patient with Cerebral Palsy. Am J Case Rep. 2015;16:469-72.
PMID: 26185982 DOI: http://dx.doi.org/10.12659/AJCR.893805 14. Gleicher N, Vidali A, Barad DH. Successful treatment of unresponsive thin endometrium. Fertil Steril. 2011;95:2123.e13-
7. PMID: 21324451 DOI: https://doi.org/10.1016/j.fertnstert.2011.01.143