1. Platelet Rich Plasma Applications in Gynecology From Basic Science to Clinical Practice Dr. Ayman Shehata Dawood Tanta University

2. History of PRP PRP has been investigated since the early 1990s and is not ‘new;’ use of autologous PRP was first used in 1987 by Ferrari et al. [Sampson, Gerhardt, Mandelbaum. Platelet rich plasma injection grafts for musculoskeletal injuries: a review. Curr Rev Musculoskelet Med. 2008 Dec;1(3-4):165-74] Today, several advancements in this innovative area of therapy are growing rapidly and gaining attraction.

3. Uses of PRP in medicine Uses of PRP in medicine PRP is now used in the following : Dermatology Plastic surgery Dentistry Orthopedics Rehabilitation & musculoskeletal, ENT Gynecology All use PRP therapy to deliver growth factors to optimize healing in their patients. In the United States, it is estimated to be used in Obstetrics & Gynecology specifically in more than 100,000 cases annually [Stammers, Trowbridge, Marko, Woods, Brindisi, Pezzuto, Klayman, Fleming, Petzold. Autologous Platelet Gel: Fad or Savoir? Do We Really Know? The Journal of ExtraCorporeal Technology. 2009;41:P25–P30].

5. Osteoarthritis Tenosynovitis

6. Platelet rich plasma Autologous Platelet-rich plasma (PRP) is derived from whole blood of the same individual, centrifuged to remove RBCs. The remaining plasma has a higher concentration of GFs 5-10 folds greater than whole blood.

7. PRP Preparation Preparation of PRP is an office procedure that involves withdrawal of blood, preparation of the PRP, and then injection into diseased area by the following steps:

8. Venous blood (15-50 ml) is withdrawn from the patient's arm in anticoagulant containing tubes. The recommended temperature during processing is 21°C-24°C to prevent platelet activation during centrifugation of blood. The blood is centrifuged at 1200 rpm for 12 minutes. The blood separates into three layers: an upper layer which contains platelets and WBC, an intermediate thin layer (the buffy coat) which rich in WBCs, and a bottom layer which contains RBCs. PRP Preparation

10. The upper and intermediate buffy layers are transferred to an empty sterile tube. The plasma is centrifuged again at 3300 rpm for 7 minutes to help in formation of soft pellets (erythrocyte-platelet) at the bottom of the tube. Discard the upper 2/3 portion of plasma as this volume is PPP (platelet-poor plasma). Pellets are homogenized in lower 1/3rd (5 ml of plasma) to create the PRP (Platelet-Rich Plasma). PRP Preparation

11. 1200 rpm 12 minutes

12. 3300 rpm 7 minutes

13. PRP Preparation PRP is now ready for injection. Nearly 30 ml of venous blood yields 3-5 ml of PRP. Clean the affected area with disinfectant before injection of PRP. Assurance and discussion with patients make the injection easier and less painful. PRP stimulates series of biological responses, and the injection site may get swollen and painful for about 3 days.

14. PRP activation Activation of platelets by the addition of thrombin, calcium chloride or collagen, resulting in fibrin polymerization and production of a gelatinous platelet gel applied to the affected site

16. PRP types After centrifugation of whole blood, four preparations can be obtained as shown in table 1. These types or classifications were proposed by Ehrenfest et al. (2009), depending on their cell content and fibrin density

17. PreparationAcronymLeucocytesFibrin density Pure Platelet-Rich Plasma P-PRPPoorLow Leucocyte- and PRP L-PRPRichLow Pure platelet-rich fibrin P-PRFPoorHigh Leucocyte- and platelet-rich fibrin L-PRFRichHigh

18. PRP composition Platelets contain high concentrations of cytokines and growth factors stored within the α -granules. More than 35 growth factors include platelet derived growth factor (PDGF), insulin like growth factor (IGF 1 & IGF 2), vascular endothelial growth factor (VEGF), platelet derived angiogenic factor (PDAF), transforming growth factor beta (TGF- β ), fibroblast growth factor (FGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF) and interleukin 8 (IL 8). In addition to growth factors (GFs), platelets contain other substances such as fibronectin, sphingosine 1- phosphate, etc…) which initiate wound healing.

19. PRP Mechanism of action The theory beyond this modality of treatment was derived from natural healing processes as the body’s first response to tissue injury is to deliver platelets to the injured area. Platelets promote healing and attract stem cells to the site of injury.

20. Growth factors promote wound healing by initiation of the following stages: Resolution, Chemotaxis, Cell regeneration, Cell proliferation and migration, Extracellular matrix synthesis, Remodeling, Angiogenesis, Epithelialization PRP Mechanism of action

21. PRP is superior to recombinant human growth factor in the release of multiple growth factors and differentiation factors upon platelet activation. Recently, the fibrin framework present over platelets was found to support regenerative matrix leading rapid morphologic and molecular configuration of wound healing. PRP ???? rHGF which is the best? PRP ???? rHGF which is the best?

22. PRP in Gynecology Though fewer articles are published on the use of autologous platelet growth factor applications to support wound healing and tissue regeneration specific to gynecology versus other clinical areas, PRP is not new to our discipline.

23. PRP uses in gynecology Surgical outcomesVulvar / Cervical lesionsUrogynecology / Genital prolapseReproductive medicineAesthetic gynecologyObstetrics

24. PRP in Surgical outcomes postoperative pain Phase I/II trial of autologous platelet tissue graft in gynecologic surgery designed to evaluate efficacy of PRP in decreasing pain was conducted on 55 patients undergoing gynecologic surgery, matched with 55 control patients. Results from this study revealed that median pain on the day of surgery was 2.7 (mild) in the autologous platelet group vs. 6.7 (severe) in the control group. Likewise, pain on postoperative day 1 was 2.1 (mild) in the autologous platelet tissue graft group vs. 5.5 (moderate) in the control group. The need for morphine during hospital stay for the autologous platelet group was 17 mg (range 1-98 mg) vs. 26 mg (range 3-90 mg) in the control group, which was statistically significant (p=0.02). Importantly, there were no adverse effects associated with autologous platelet, and patient pain was significantly reduced

25. Tehranian et al, (2016) tested PRP in wound healing of high- risk women undergoing cesarean sections. They applied PRP in 70 patients and compared them with 71 control cases without PRP application. The inclusion criteria were body mass index (BMI) > 25, prior cesarean section, diabetes, twin pregnancy, use of corticosteroid medication and anemia. They found that greater reduction in the edema, ecchymosis and discharge with better approximation (REEDA) score compared to the control group (85.5% reduction in the PRP group; 72% in the control group) (P < 0.001). They concluded that PRP is an effective therapeutic approach for wound healing, and faster wound healing is expected due to the presence of more platelets and growth factors. PRP in Surgical outcomes wound healing

26. Morelli et al, (2013) conducted a retrospective study to evaluate the efficacy of platelet gel application on healing after radical vulvectomy. They found significant decrease in wound infection (P = 0.032), necrosis of vaginal wounds (P = 0.096), and breakdown of wound (P = 0.048) rates in PRP group compared to control group. They also found that reduction in postoperative fever rate, hospital stay, and faster wound healing were also detected in group A treated by PRP gel. They concluded that platelet gel application before vulvar reconstruction represents an effective strategy to prevent wound breakdown after local advanced vulvar cancer surgery. PRP in Surgical outcomes wound healing

27. PRP uses in gynecology Surgical outcomesVulvar / Cervical lesionsUrogynecology / Genital prolapseReproductive medicineAesthetic gynecologyObstetrics √√

28. PRP in Vulvar / Cervical lesions Vulvar dystrophies PRP was tested in many dermatological and autoimmune conditions not responsive to corticosteroids such as Lichen sclerosus (LS) and eczema. Lichen sclerosis affects vulva and causes extensive scarring with progressive loss of the labia minora, sealing of the clitoral hood, and burying of the clitoris. Lichen sclerosus causes progressive pruritus, dyspareunia, or genital bleeding. LS has a considerable impact on quality of life of affected patients by disturbing physical activity, sexual satisfaction, and causing emotional and psychological effects. This condition is treated by topical and systemic corticosteroids.

29. Application of PRP in resistant cases of Lichen sclerosus to steroid therapy was conducted by Willison et al, (2016) in 28 patients with LS. They injected PRP in vulva in fanning pattern. Patients received 3 PRP treatments 4 to 6 weeks apart and again at 12 months. They found that nearly all patients exhibited clinical improvement in the size of their lesions and in 28.6% of patients lesions disappeared completely after treatment with PRP. Minimal pain and zero complications also were reported. They concluded that PRP injection of PRP can therefore be considered effective therapy for LS. PRP in Vulvar / Cervical lesions Vulvar dystrophies

30. Hua et al, (2102) conducted a randomized clinical study to compare the effectiveness of autologous platelet-rich plasma (PRP) applications to laser in the treatment of benign cervical ectopy. They applied RPP twice on the cervical erosion with one week interval in 60 patients while Laser was applied in the other 60 patients. They found that the complete cure rates were 93.7% for the PRP and 92.4% for the laser group (P>0.05). The mean time to re- epithelialization was significantly shorter in the PRP (P<0.01). The rate of adverse treatment effects (i.e. vaginal discharge or vaginal bleeding) was much lower in the PRP than in laser group (P<0.01). They concluded that autologous PRP applications appear promising for the treatment of cervical ectopy in symptomatic women, as they generate a shorter tissue healing time and milder adverse effects than laser treatment. PRP in Vulvar / Cervical lesions Cervical ectopy

31. PRP uses in gynecology Surgical outcomesVulvar / Cervical lesionsUrogynecology / Genital prolapseReproductive medicineAesthetic gynecologyObstetrics √√

32. Shirvan et al, (2013) in 12 patients injected platelet rich plasma around the fistula into the tissue and platelet rich fibrin glue was interpositioned in the tract. They followed cases for 6-months and found that 11 patients become clinically cured, and transvaginal physical examination and cystography were normal. They concluded that Autologous platelet rich plasma injection and platelet rich fibrin glue interposition offer a safe, effective and novel minimally invasive approach for the treatment of vesicovaginal fistula which obviate the need for open surgery.

33. Gottgens et al, (2015) used PRP injection into fistula tract after mucosal advancement flap in 10 patients with Crohn’s disease-related high perianal fistulas. They found that healing of the fistula was 70 % at 1 year. One patient (10%) had a recurrence, and in two (20 %) patients, the fistula was persistent after treatment. They concluded that results of this procedure in crohn's disease fistulae are moderate with success rate of 70% and further studies are needed.

34. Bodner-Adler et al, (2017) conducted a systematic review on genital fistulae treatment where conservative and surgical treatments were assessed. They found that small fistulae could be treated conservatively with different therapies including PRP with success rate ranged between 67%-100%. They concluded that PRP is tried in treatment of vesicovaginal fistula fistulae as a novel minimally invasive approach for closure of genital fistulae.

35. Various types of vaginal implants, absorbable and non-absorbable, that have been introduced in pelvic floor reconstructive surgeries have numerous serious adverse effects. Platelet rich fibrin (PRF) based on stimulation of fibroblast migration and proliferation was used in vaginal surgery for rapid remodeling and connective tissue growth. Genital prolapse

36. Gorlero et al, 2012 conducted study on 10 consecutive women requiring surgery for prolapse recurrence (stage II or higher). They operated cases plus injection of PRF. They found that the success rate was 80% with complete symptom relief. Sexual activity increased by 20% without dyspareunia. They concluded that the use of PRF at site-specific prolapse repair is associated with a good functional outcome. On the same hand, Medel et al, (2015) found that attachment of fibroblast to vaginal meshes was significantly increased after coating meshes with PRP in-vitro.

37. On the other hand, another study was conducted to evaluate whether autologous platelet gel application during anterior colporrhaphy increases collagen content of the pubocervical fascia and creates more durable repair. The authors applied autologous platelet gel to the surgical site during anterior colporrhaphy in 9 patients. Biopsy specimens from the anterior vaginal wall at surgery and 3 months postoperatively were collected. They found no significant increase occurred in collagen content at 3 months after operation and they concluded that autologous platelet gel didn't increase collagen or durability of the repair.

38. Recently Chrysanthopoulou et al, (2017) summarized the existing evidence based on animal experimental and clinical studies that address the potential role of PRP in treating genital prolapse. They concluded that PRP restore the anatomy and function of pelvic ligaments but up till now there is no evidence to support or oppose PRP use in women suffering from genital prolapse.

39. Nikolopoulos et al, (2016) summarized studies advocating the use of PRP in urinary incontinence resulting from damage to the pubourethral ligament. They found that PRP helps in regulating tissue reconstruction and restoration of pubourethral ligament strength; but studies were not giving sufficient evidence to validate its use. PRP in urogynecology and genital prolapse Urinary incontinence

41. PRP uses in gynecology Surgical outcomesVulvar / Cervical lesionsUrogynecology / Genital prolapseReproductive medicineAesthetic gynecologyObstetrics √√

42. PRP in reproductive medicine Premature ovarian failure Premature ovarian failure (POF) is a loss of normal function of the ovaries before age 40 with loss of fertility. A team of Harvard University researchers changed this fact when they injected mice's ovary with growth factors and appeared to develop mature eggs from ovarian stem cells. They stated that introduction of isolated growth factor- bearing platelets directly into the ovaries might trigger a resurgence in oocyte production.

43. PRP in reproductive medicine Premature ovarian failure PRP therapy is recommended in women with premature ovarian failure (POF), infertile women >35 years and women with low ovarian reserve. Treatment with PRP is named ovarian rejuvenation where PRP is injected into the ovary by ultrasound guidance like ovum retrieval in IVF. This modality of treatment is still under trials. Pantos et al, (2016) in the ESHRE annual conference held in 2016 at Helsinki, Finland introduced this modality (Ovarian rejuvenation). They injected PRP in 8 peri-menopausal/POF women with poor ovarian reserve. They found successful ovarian rejuvenation 1-3 months after PRP treatment. All cases undergone natural IVF cycles with resulting follicle of 15.20±2.05 mm in diameter and the resulting oocytes were inseminated by ICSI and all resulted embryos were cryopreserved.

44. 32nd Annual Meeting of ESHRE, Helsinki, Finland, 3 July – 6 July, 2016.

45. Ovarian rejuvenation registered on clinicaltrials.gov 2017 Autologous Platelet-Rich Plasma (PRP) Infusions and Biomarkers of Ovarian rejuvenation and Aging Mitigation

46. PRP in reproductive medicine Refractory Endometrium The endometrium is an important factor involved in achieving optimal outcomes after assisted reproductive treatment. Endometrial growth following inadequate ovarian stimulation may be neglected leading to poor results of IVF/ICSI cycles. Different strategies were suggested to improve endometrial thickness especially in resistant cases. PRP is one of those therapies tried in patients with refractory endometrium.

47. PRP in reproductive medicine Refractory Endometrium Colombo et al, (2017) included 8 patients to undergo PRP treatment. The inclusion criteria were women with more than 3 cancelled cryo-transfers due to poor endometrial growth< 6 mm, women with negative hysteroscopic screening for endometrial pathology, and women with negative bacteriologic screening. After application of PRP, the endometrial thickness was satisfactory in 7 cases. Of these, beta-HCG was positive in 6 women. They concluded that the multiple implantation failures were caused by inefficient expression of adhesion molecules, which can hypothetically be more represented after PRP application.

48. PRP in reproductive medicine Refractory Endometrium Zadehmodarres et al, (2017) in their pilot study include 10 patients who had a history of cancelled cycles due to inadequate endometrial growth (less than 7 mm). They found that endometrial thickness increased at 48 hours 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. Five patients were pregnant (50%) and in four of them the pregnancy progressed normally. They concluded that PRP was effective for endometrial growth in patients with thin endometrium.

49. Jang et al, (2017) in animal model investigated the role of PRP in regeneration of endometrium after ethanol induced damage. They found that intrauterine administration of autologous PRP stimulated and accelerated regeneration of the endometrium and also decreased fibrosis in animal model of damaged endometrium. PRP in reproductive medicine Refractory Endometrium

50. Yajie Chang, 2015, evaluated the effectiveness of PRP in management of infertile women with thin endometrium (≤ 7 mm) in thawed embryo transfer. They included 5 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. 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. PRP in reproductive medicine Refractory endometrium

51. PRP in reproductive medicine Repeated implantation failure Repeated implantation failure (RIF) is defined as failure to conceive following several embryo transfers in IVF cycles. Numerous factors are involved in process of implantation including embryo quality, endometrial receptivity and immunological factors. Several measures were suggested for RIF management but there is little consensus on the most effective one. These measures include blastocyst transfer, assisted hatching, hysteroscopy, endometrial scratching, and immune therapy. Recently, intrauterine infusion of platelet-rich plasma (PRP) is described to promote endometrial growth and receptivity.

52. Nazari et al, (2016) enrolled 20 participants with history of RIF into their study to evaluate the effectiveness of platelet-rich plasma in improvement of pregnancy rate in RIF patients. The inclusion criteria were below 40 years and their body mass index (BMI) below 30 kg/m 2. They found that 18/20 (90%) of participants got pregnant. Sixteen clinical pregnancies were recorded and their pregnancies are ongoing. They concluded that PRP is effective in improvement of pregnancy outcome in RIF patients. PRP in reproductive medicine Repeated implantation failure

53. Clinicaltrial.gov

55. PRP before thawed embryo transfer

56. PRP uses in gynecology Surgical outcomesVulvar / Cervical lesionsUrogynecology / Genital prolapseReproductive medicineAesthetic gynecologyObstetrics √√

57. PRP in Aesthetic gynecology Breast reconstruction Gentile et al, (2013) enrolled 100 patients aged between 19 and 60 years affected by breast soft-tissue defects. They divided patients into 2 groups with equal allocation; the study group was treated with fat grafting + PRP. The control group was treated with fat grafting injection only. They found that patients treated with PRP added to the autologous fat grafts showed a 69% maintenance of the contour restoring and of 3-dimensional volume after 1 year, whereas the patients of the control group treated with centrifuged fat grafting showed a 39% maintenance. They concluded that PRP mixed with fat grafting leads to an improvement in maintaining breast volume in patients affected by breast soft-tissue defects.

58. PRP in Aesthetic gynecology Female sexual dysfunction Medical professionals know that platelets release around 35 growth factors that promote tissue regrowth, healing, and regeneration. This fact was utilized by aesthetic gynecologists in many aspects including vaginal rejuvenation and O-shot therapy.

59. PRP use in sexual dysfunction is considered a new non- surgical office treatment that helps improve both urinary incontinence and sexual dysfunction through using woman's own growth factors. The PRP is injected into specific areas of the vagina with the aid of local anesthetic cream. This modality of treatment is called "o-shot". PRP immediately activate tissue regeneration and the enhancement in sexual response is dramatic. The desired response includes improved arousal, stronger orgasm, decreased dyspareunia, and increased natural lubrication. PRP in female sexual dysfunction O-Shot therapy

60. PRP in Aesthetic gynecology Female sexual dysfunction Runels et al, (2014) enrolled 11 females presenting with dyspareunia in their study. They injected PRP into clitoris and vagina. They found that intravaginal and intraclitoral PRP injections could be an effective method to treat certain types of female sexual dysfunction, especially in the areas of desire, arousal, lubrication and orgasm.

63. PRP in Aesthetic gynecology PRP in Aesthetic gynecology Vaginal rejuvination PRP is used in regeneration of vaginal mucosa, muscles and skin. After PRP injection, vaginal vascularity is increased with subsequent dramatic increase in sensitivity. In addition, the skin becomes thicker and firmer, making vagina looks much more youthful. More over ligaments and muscles supporting urethera, become more stronger alleviating urinary incontinence.

64. PRP in Aesthetic gynecology PRP in Aesthetic gynecology Vaginal rejuvenation Kim et al, (2017) reported the use of PRP in one case for vaginal rejuvenation. They concluded that application of autologous lipofilling mixed with PRP in vaginal atrophy produced relief of symptoms, contour restoration. The rejuvenated appearance of external genitalia provided pleasing cosmetic outcome to the patient.

68. PRP uses in gynecology Surgical outcomesVulvar / Cervical lesionsUrogynecology / Genital prolapseReproductive medicineAesthetic gynecologyObstetrics √√

69. PRP in Obstetrics Premature rupture of membranes (PROM) occurs due to damage and tears in the fetal membrane leading to congenital infections and poor neonatal outcomes. PRP was tried in-vitro model to evaluate the ability of platelet-rich plasma (PRP) in sealing the iatrogenic fetal membrane defect. This was done on single and double layers amnion models. The PRP plug was stable and attached firmly to amnion tear. Authors concluded that there is experimental evidence that a PRP plug persists for nearly 2 months in an amniotic fluid environment. It also provides waterproof sealing of iatrogenic defects in amnion and chorion. Moreover PRP stimulates cell growth and proliferation and may thereby enhance a membrane healing response.

71. Conclusions Platelet rich plasma is an innovative therapeutic modality being cheap, simple, easily commenced, safe and effective. It was tried in many fields of medicine and proved effective. In gynecology studies are still few, pilot or case series. Large randomized controlled studies are required for approval of the efficacy and safety of Platelet rich plasma in gynecologic disorders.