Autologous platelet-rich plasma (PRP) treatment of ovarian tissues
PRP ovarian rejuvenation was performed on the 23rd of November 2015. The FSH, E2, and LH levels before the procedure were 4.99 mIU/mL, 421.7 pg/mL, and 10.26 mIU/mL, respectively. Briefly, 120 mL of whole blood were withdrawn from the cubital vein and anticoagulated using acid citrate dextrose formula A (ACD-a) in a 7:1 ratio. Whole blood was centrifuged using a tabletop blood separation system, and 7 mL of PRP were obtained. After centrifugation, the concentration of platelets was 6.5 × baseline with leucocytes 0.6 × baseline. PRP was activated using autologous thrombin at a ratio of 10:1. After the activation, the volume of PRP (6 mL) was instilled into the ovaries via transvaginal, ultrasound-guided injection using a 17G needle under total anesthesia, 2 mL to the left, and 4 mL to the right ovary. After needle priming, the obtained volume of PRP was injected as multiple subcortical injections, in volumes between 0.5 to 1 mL in five positions per ovary. The patient suffered no blood loss during the procedure, recovered and was discharged from the hospital the next day.
The patient had irregular menstrual cycles following the procedure, and no follicular growth was observed in monthly ultrasound examinations during the next six months. She continued treatment with Cyclo-Progynova (0.5 mg/2 mg; 2 mg, norgestrel/estradiolvalerat, BAYER WEIMAR GMBH & CO.KG) and Estrofem (2 mg estradiol, Novo Nordisk, Denmark) during that period. At that time, the patient decided to undergo a SEGO procedure, which is an upgraded rejuvenation method compared to PRP rejuvenation.
SEGO ovarian rejuvenation: autologous platelet-rich plasma (PRP) and autologous bone marrow stem cell ovarian injection
In June 2016, the patient had FSH, E2, LH, and AMH levels of 11.2 mIU/mL, 391 pg/mL, 2.9 mIU/mL, and < 0.2 ng/mL, respectively. The SEGO procedure was performed on 13th July 2016. PRP preparation was performed as described in section 3.1. A total of 5 mL of activated PRP were obtained for the treatment. Bone marrow (BM) sampling from the tibial bone was performed under general anesthesia, and a small incision (7 mm) was made to penetrate the periosteum. A total of 80 mL of bone marrow was aspirated and centrifuged under sterile conditions, and three layers were obtained: the acellular portion (platelet-poor plasma, PPP), the cells (RBC), and the bone marrow aspirate concentrate (BMAC) containing nucleated cells. The obtained BMAC (3 mL) was diluted to 4 mL with PPP and further used for treatment. Afterwards the procedure, flow cytometry was used to determine the total nucleated cell count (TNC), which was 36.7 × 106 cells/mL, and the cell viability was 96%.
The prepared PRP and BMAC were instilled together into the ovaries via transvaginal, ultrasound-guided injection using a syringe system and a 17G needle, which was performed under total anesthesia. After needle priming, the obtained volume of 3 mL of PRP and 2.5 mL of BMAC were injected into the right ovary, while 2 mL of PRP and 1 mL of BMAC were injected into the left ovary, as multiple subcortical injections, in volumes between 0.5 to 1 mL in five positions per ovary. The patient suffered no blood loss during the procedure, and she recovered and was discharged from the hospital the next day.
One month after the procedure, the patient underwent the first control examination and reported no adverse effects or complications. Ultrasound control performed on 15th August 2016, reported normal menstrual cycling (28/3 days), the endometrium in the periovulatory phase, and an endometrial thickness of 8 mm on the 11th day of the menstrual cycle. Several follicles smaller than 4 mm were observed on the left ovary. The patient continued to receive Cyclo-Progynova (0.5 mg/2 mg; 2 mg, norgestrel/estradiolvalerat, BAYER WEIMAR GMBH & CO.KG), and Estrofem (2 mg estradiol, Novo Nordisk, Denmark) therapy during the next six months. Until January 2017, the patient had regular menstrual cycles with follicles growing up to 10 mm in diameter. In February 2017, the patient was suggested to undergo an advanced intervention of ovarian rejuvenation called SEGOVA, which will be described in the next section.
SEGOVA ovarian rejuvenation: ovarian cortical tissue resection, in vitro tissue activation with autologous PRP and BMAC transplantation to the ovaries
In February 2017, a 33-year-old patient had the following hormonal levels before the procedure: FSH, 4.23 mIU/mL; E2, 18.17 pg/mL; LH, 1.08 mIU/mL; and, Beta Human Chorionic Gonadotropin hormone (Beta-hCG) 0.1 mIU/mL. On 10th February 2017, the patient underwent SEGOVA procedure. Before laparoscopy, PRP preparation was performed. Briefly, whole blood was withdrawn from the cubital vein and was anticoagulated using ACD in a 7:1 ratio. Two 60 mL syringes were used to obtain a total volume of 104 mL of whole blood and 16 mL of ACD-a. Next, the blood was centrifuged using a tabletop blood separation system. After separation, the layers were composed of approximately 53 mL of RBC, 4 mL of PRP, and 47 mL of PPP. Next, platelet count was determined and the PRP was diluted up to 5 mL using PPP. Autologous thrombin was used for activation. Activation was performed in order to release growth factors from the platelets. PRP was activated using autologous thrombin at a ratio of 10:1. Activated PRP (5 mL) was further used for tissue treatment.
Laparoscopic resection of ovarian cortex was then performed using a standard procedure, using a laparoscopic technique with an entry through the umbilicus. After making an incision of approximately 2 cm in the umbilical zone, three portals of 5 mm were placed and intra-abdominal pressure between 10 and 12 mmHg was established. A laparoscope of 5 mm in diameter was then introduced, together with auxiliary trocars. After visualization of the ovaries using an adequate instrument, the cortex was fixed, and scissors were used to cut off a part of the cortical tissue. The cortical tissue measured was 254 mg. Hemostasis, port performance, and wound closure were checked. During the further course of the process, the ovary cortex obtained via multiple cutting with scalpel No. 25 was cut into fragments smaller than 1 × 1 mm2. The tissue prepared in this manner was measured using an analytical scale and placed on a Petri dish and washed using the gammet buffer (Sydney IVF gamete buffer, Cook Medical, USA). After rinsing, the tissue samples were transferred to PRP media and continued to be activated via autologous thrombin mediation. The prepared sample was incubated with the volume of activated PRP (5 mL) for 48 h at 37°C and 5.5% CO2.
Bone marrow sampling
After 48 h, bone marrow sampling from the proximal tibia was used to obtain stem cells. Biopsy was performed under general anesthesia, and a small cut (7 mm) was used to penetrate the periosteum. Bone marrow (95 mL) was aspirated and centrifuged using a specially automated system under sterile conditions. After separation, the acellular portion and the RBCs were discarded, while 4 mL of BMAC with nucleated cells were further used for the treatment. In addition, 1 mL of the BM and BMAC sample was used to evaluate cell viability, the TNC, and to perform flow cytometry phenotypic characterization of stem cells from BMAC using a set of specific markers: (CD73, CD90, CD105, CD133, CD 271). Figure 1. presents analyzed CD markers before and after BM concentration. For BMAC the determined TNC was 27 × 106 cells/mL, and the viability was 98.7%.
After BMAC was obtained and ovarian cortex tissue was incubated for 48 h after the the fragmented tissue of the ovary with 5 mL of PRP was injected into the subcortical region of the right and left ovaries, together with 4 mL of BMAC, under the ultrasonic monitoring of 3D color ultrasound GE Voluson 730 Pro, via a transvaginal puncture under general anesthesia using a 16G needle. Injection of PRP, tissue fragments, and BMAC in each ovary was performed as multiple subcortical injections, in volumes between 0.5 and 1 mL in five positions per ovary. The procedure did not involve any complications, and there was no blood loss. The patient was discharged from the hospital the day after the procedure.
All procedures performed in this study were approved by the Institutional Board of Ethics at Special Gynecology Hospital Jevremova, Medigroup, Belgrade, Serbia (IRB No 63/295/2015) and in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. An informed consent was obtained from the participant included in the study.
One month after the procedure, on 9th March 2017, follicular growth was observed and the patient received a short protocol of stimulation with Menopur (menotrofin 75 IU, Ferring GMBH) and Ovitrelle (250 mcg/0.5 mL injection, Merck). On the 15th day of the menstrual cycle, (16th March 2017), the E2 level was 102 pg/mL, the LH level was 31.65 mIU/mL, and the follicles were punctured without retrieving any eggs. In May 2017, the cycle was stimulated with Merional (menotrofin 75 IU, IBSA Institut Biochimique S. A.) and the patient received Klomifen (Clomiphene 50 mg, REMEDICA Ltd., Cyprus). At the end of the stimulation protocol, the E2 level was 65.84 pg/mL and no follicle growth was detected. During June 2017, the patient received a stimulation protocol with Pergoveris (150 IU/75 IU powder and a solvent for injection of Follitropin alfa/Lutropin alfa, Merck Serono Gmbh, but with no follicular growth, and the E2 level was 53.73 pg/mL at the end of the stimulation. In September 2017, the patient had regular menstrual cycles under Cyclo-Progynova therapy (0.5 mg/2 mg; 2 mg, norgestrel/estradiolvalerat, BAYER WEIMAR GMBH & CO.KG) and Estrofem (2 mg estradiol, Novo Nordisk, Denmark). Natural IVF was planned, instead of a stimulated IVF cycle. On the 6th October 2017 (eight months after the procedure), on the 10th day of the menstrual cycle, the endometrial thickness was 7 mm when follicle growth was detected. The patient presented with a 14-mm diameter follicle on the right ovary. On the next day, (7th October 2017), several larger follicles were aspirated, and one Metaphase II (MII) oocyte was obtained. The hormonal levels were as follows: LH, 49.42 mIU/mL; E2, 114.8 pg/mL; and PG, 0.864 ng/mL. Intracytoplasmic sperm injection (ICSI) was performed, but total fertilization failure occurred. After that, the patient did not undergo any further treatments.
Three months later, in January 2018, the patient reported to be in the 11th week of spontaneous pregnancy. The patient also reported that she was not on therapy at the time of conception. The pregnancy went to term without complications. Birth occurred spontaneously via uncomplicated vaginal delivery, and the Apgar score of the newborn was 9/10.