Autologous ovarian platelet rich plasma treatment improves oocyte and embryo quality: a before-after prospective study

Abstract Platelet-rich plasma (PRP) is used for successful regeneration of female reproductive tissues. However, little is known about the effect of ovarian PRP treatment on oocyte and embryo quality. The objective of our study was to assess the role of autologous ovarian PRP treatment on ovarian reserve and number and quality of oocytes and embryos in women with poor ovarian response (POR) undergoing in-vitro fertilization cycles. A total of 66 women with POR were treated with ovarian PRP injection in two subsequent menstrual cycles. The antral follicle counts, serum anti-Mullerian hormone, follicle-stimulating hormone levels, fertilization rate, number and quality of oocytes, and embryos were assessed and compared between the cycle before and after PRP treatment. Ovarian PRP treatment resulted in insignificantly lower follicle-stimulating hormone levels, significantly higher antral follicle count, anti-Mullerian hormone, number of retrieved oocytes, and insignificantly higher fertilization rate. However, the mean number of Day 5 embryos (2.19 ± 1.45 vs. 1.58 ± 1.30, p = 0.01), the percentage of high-quality oocytes (45.29% ± 42.40% vs. 15.21% ± 30.24%, p < 0.01) and the percentage of grade-I blastocysts (52.10% ± 37.94% vs. 12.86% ± 22.97%, p < 0.01) were significantly higher after PRP treatment in comparison to the pretreatment period. Moreover, the mean MII oocyte quality (1.60 ± 0.54 vs. 2.31 ± 0.63, p < 0.01) and mean blastocyst quality (1.53 ± 0.45 vs. 2.42 ± 0.63, p < 0.01) were significantly improved in the post-treatment period. In conclusion, the applied autologous ovarian PRP treatment in poor responders may significantly improve oocyte and embryo quality.


Introduction
A crucial aspect of successful in-vitro fertilization (IVF) is the yield of a sufficient number of oocytes suitable for fertilization. IVF outcome is largely dependent upon the specific patient response to ovarian stimulation, as it is highly associated with the number and quality of the retrieved oocytes. Between 9% and 24% of all IVF cycles did not respond adequately to standard ovarian stimulation protocols, as a result of poor ovarian response (POR) to the performed hormonal stimulation [1]. Presently, treatment of POR patients is still a challenging task [2].
Platelet-rich plasma (PRP) is defined as plasma containing increased concentration of platelets several-fold over that of the plasma base level [3]. Platelets contain high levels of integrins, cytokines, chemokines and growth factors [4] that promote cell differentiation, migration, angiogenesis, tissue restoration and repair [5,6]. Recently, many human and animal studies have shown a beneficial impact of PRP on infertile subjects based on the existing intrinsic regenerative mechanisms [7][8][9]. During the last few years PRP has become widely used in regenerative medicine in patients with unexplained implantation failures and thin endometrium but also in cases with diminished ovarian reserve and ovarian failure [3,4,[7][8][9][10][11][12][13]. However, the exact mechanisms are not fully understood and the applied techniques and protocols for an optimal PRP production and an adequate treatment are still under development. Although the autologous ovarian PRP injection has already been used [12,13], the exact effect of this treatment on the oocyte and embryo quality is scarcely investigated.
The aim of the present before-after prospective study was to evaluate the effect of calcium gluconate-activated autologous ovarian PRP on ovarian reserve and oocyte and embryo quantity and quality. Our main outcome measures were antral follicle count (AFC), follicle-stimulating hormone (FSH) anti-Mullerian hormone (AMH), fertilization rate, number and quality of oocytes and embryos.

Ethical approval
The approval of the study protocol was granted by the local Ethics Committee (No: 02/21.01.2019). To ensure anonymity, patients' information was de-identified. Verbal informed consent was obtained from each patient and her partner. All 66 women and their partners agreed to participate in this study.

Patient selection and experimental design
The study was performed at Nadezhda Women's Health Hospital, Sofia, Bulgaria between March 2021 and September 2021. During the study period 71 women were eligible for enrolment ( Figure 1) and finally sixty-six patients with POR and more than one ICSI cycle were analyzed in this before-after prospective study ( Figure 2). The Bologna criteria were used to define PORs [14]. The inclusion criteria for trial NCT04797377 were: a previous assisted reproductive technology cycle with less than 3 oocytes (conventional stimulation protocol) and AFC < 7. Additional inclusion criteria were: the same ovarian stimulation protocol before and after the PRP treatment, and to have a standard ICSI cycle followed by PRP ovarian treatment and a second ICSI cycle. Exclusion criteria for the study group were age over 46 years, body mass index (BMI) ≥ 30 kg/m 2 , presence of pregnancy, uncontrolled endocrine disorders (polycystic ovary syndrome and others), parental genetic and chromosomal disorders, immunological disorders and cancer diagnostics. Data collected include women's baseline characteristics (age, BMI, serum AMH and basal FSH levels), AFC, number and quality of oocytes retrieved, fertilization rate, and quantity and quality of embryos available. All recorded and studied variables from one IVF cycle of each participant before PRP treatment were compared to the same variables from the first cycle following PRP treatment (<3 months after the performed PRP treatment).

Controlled ovarian stimulation, oocyte retrieval, ICSI and embryo culture
All women were stimulated according to a standard long protocol starting with a pituitary downregulation with a gonadotropin-releasing hormone analogue (Decapeptyl, Ferring, Switzerland) (3.75 mg) followed by administration of recombinant follicle stimulating hormone (Gonal, Merck Serono Ltd, uK) (300 Iu/day). Follicular growth was monitored by ultrasound at day 4 after gonadotropin stimulation and every other day if indicated. Ovulation was triggered by injection with human chorionic gonadotropin (5000 Iu) (Choriomon, IBSA, Lugano, Switzerland) once one or more follicles reached 18 mm diameter. Oocytes were retrieved up to 36 h after the trigger under transvaginal ultrasound guidance.
In all cases, oocytes were fertilized by ICSI in 4-6 h after retrieval and cultured separately under mineral oil, in 20-µL droplets of single-step medium (Global Total, IVFonline, Guelph, ON, Canada) in 6% CO 2 and 5% O 2 atmosphere at 37 °C. Fertilization status of the oocytes was reported in 16-18 h after ICSI according to the presence of two pronuclei. Fertilization rate was calculated as the percentage of transformation of the injected oocytes into two pronuclei.

Hormonal measurements
Blood samples for detection of AMH and basal FSH were collected before and after PRP injections by venipuncture on day 2 to day 4 of the menstrual cycle. Serum AMH and FSH concentrations were determined by an electrochemiluminescent (ECLIA) immunoanalyzer (Cobas e411, Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturers' instructions. The sensitivity detection limit for FSH and AMH were 0.1 mIu/mL and 0.01 ng/mL, respectively.

Sample preparation for PRP
Approximately 22 mL of blood was collected from each patient by peripheral venipuncture using a 21 G butterfly catheter affixed via vacutainer to negative pressure receiving tubes (BD vacutainer acid-citrate-dextrose (ACD-A), BD-Plymouth, uK, REF:366645). Samples were spun in a room-temperature centrifuge set at 500×g for 5 min. After centrifugation, the supernatant containing the PRP fraction was aspirated and centrifuged at 700×g for 7 min. The supernatant was aspirated and the platelets were resuspended in 1 ml of plasma.

PRP activation and intraovarian injection
The PRP was activated by addition of calcium gluconate as described previously [9,15]. Briefly, the calcium gluconate and the PRP were mixed in a 1:9 ratio (0.05 mL calcium gluconate per 0.5 mL of PRP). The activated PRP samples were divided into two syringes (2 × 0.5 mL)-one for each ovary-and 11.8 inch (30 cm) single lumen 21 G needles were attached. Prepared activated PRP samples were maintained at room temperature until application.
The PRP injection was performed under general anesthesia. The ovaries were reached using needle guide preventing vascular or other structures rupture. The needle was advanced into the center of the ovarian medula without rotation. The correct tip placement was confirmed by ultrasound. The activated PRP was slowly introduced during careful retraction of the needle across the previously traversed ovarian cortex. The final PRP volume was deposited under the ovarian capsule and the needle exit the ovary [16]. The described procedures followed the trial NCT04797377 protocol.
The PRP was applied once between day 7 and day 11 of the menstrual cycle for two consecutive cycles. The decision to undertake two treatment cycles was based on that the antral follicle development takes from 90 to 120 days. We suggested that repeated platelet stimulation would have an optimal effect on follicle growth and oocyte maturation. No complications related to the applied procedure were reported.

Measurement of blood platelet concentration
Blood cell counts (platelets, red blood cells and white blood cells) were obtained using an XN-1000 analyzer (Sysmex, Japan). The platelets were measured in units × 10 6 /µL. All samples were collected into cryovial tubes, tested within 2 h of preparation. quality control (qC) of the analyzers was performed with three levels of XN-Check material (Sysmex, Japan).

Oocyte quality evaluation
The oocytes were classified using a previously described protocol based on morphological assessment and enumeration of extracytoplasmic anomalies (rough or fragmented first polar body and/or a large perivitelline space) and intracytoplasmic anomalies (dark cytoplasm, granular cytoplasm or refractile bodies) [17]. The oocytes were graded as excellent (1), good (2) and poor quality (3) according to the number of defects observed: grade 1: oocytes without any anomaly; grade 2: oocytes with one anomaly and grade 3: oocytes with at least two anomalies.

Embryo quality evaluation
Morphological evaluation of the cultured embryos was conducted 120 or 144 h following sperm injection. Blastocysts scoring was performed on day 5 or 6 depending on the blastocoel cavity expansion and on the inner cell mass and trophectoderm cells integrity [18]. The included scoring variables that were evaluated were: cell number, symmetry and granularity, type and percentage of fragmentation, presence of multinucleated blastomeres and degree of compaction. The grading scores applied were: 1 (excellent quality: regular blastomeres, without fragments), 2 (moderate quality: irregular blastomeres, without fragments), and 3 (poor quality: irregular blastomers, with fragments).

Statistical analysis
Patient data were analyzed with SPSS v.21 (IBM, Armonk, Ny). Comparisons across means were evaluated by Wilcoxon signed-rank test. Differences were considered statistically significant at the p < 0.05 level.

Baseline characteristics and PRP characteristics
The patients' baseline characteristics are presented in Table 1. The age of the women was 30-46 years and they had a body mass index between 19 and 27 kg/ m 2 . The second IVF cycle was on average 46 days after the second intraovarian PRP application. The main characteristics of the obtained and subsequently applied PRP throughout the study are shown in Table 2. The final concentration rates of platelets in the prepared PRP fractions were 6.2-fold higher than those in the whole blood samples. Both the red blood cell count and the white blood cell count were reduced by 230.6-fold and 2.8-fold, respectively and platelets were concentrated 6.2-fold.

Ovarian reserve, oocyte and embryo quality (IVF characteristics)
Basal FSH levels measured on day 3 of the cycle were not significantly changed after PRP treatment, compared to the basal FSH levels before PRP (Table 3). A significant increase in serum AMH was found in the studied patients after PRP treatment. The average number of mature follicles was slightly higher after PRP treatment, but not significantly different when compared to the pretreatment period. Тhe mean fertilization rate of MII oocytes was also not significantly different after PRP treatment (Table 3).
In contrast, the number of retrieved metaphase II (MII) oocytes, the mean number of Day 5 embryos, the percentage of high-quality MII oocytes and the percentage of grade-I blastocysts were significantly higher after PRP treatment in comparison to the pretreatment period (Table 3; Figure 3). In addition, the mean MII oocyte quality (1.60 ± 0.54 vs. 2.31 ± 0.63, p < 0.01) and the blastocyst quality (52.10% ± 37.94% vs. 12.86% ± 22.97%, p < 0.01) were significantly improved in the post-treatment period. Finally, we observed improvement in the mean quality of oocytes in 78% of the women and improvement in the mean quality of blastocysts in 86% of all patients.

Discussion
In this before-after prospective study, we were focused to examine the impact of PRP ovarian treatment on oocyte and embryo quality. The main factors that could have an essential influence on embryo quality and confounding effect on the final results were reduced by comparing the selected outcome variables before and after PRP ovarian treatment from IVF cycles in the same women using the same stimulation protocol. A limitation to our study is the use of visual evaluation of the oocytes and the embryos. Although we applied standard morphological characteristics for evaluating oocyte and embryo quality, these are not always strongly associated with the implantation success [19]. Therefore, by only using these qualitative descriptions, we are aware that crucial comparative data might be missing. We found out that 78% of women have better oocyte quality and 86% have improved embryo quality after PRP treatment. It is still possible that the oocyte and embryo quality are improved in an even higher percentage of the poor responders following PRP treatment, but our ability to detect and measure that quality could be defined as our limiting factor.
Previous studies have revealed that the most effective therapeutic effect of PRP could be observed if the growth factors and cytokines are injected directly into the ovarian tissue [20]. The role of PRP sample activation by addition of calcium gluconate is also an Table 2. Descriptive statistics of pRp characteristics. characteristics mean* (min-max) platelet count, 10 6 /µl 1.7 (1-2.1) WBc count, 10 6 /µl 0.9 (0.1-2.2) RBc count, 10 9 /µl 0.02 (0.01-0.06) concentration rates of platelets 6.2 (4.9-7.3) pRp, quantity per dose, ml 0.5 activation, ml ca gluconate 0.05 * the variables are presented as mean (range) or number only. WBc, white blood cells; RBc, red blood cells; pRp, platelet-rich plasma. important step because it facilitates the growth factor release [13,20].
In the present prospective study, the quality of retrieved oocytes was significantly higher after treatment with PRP. In addition, an increase in the number of MII oocytes was observed. Also, PRP therapy has resulted in improved embryo quality and a significant increase in the number of high quality blastocysts, which allowed better embryo selection, higher number of successfully vitrified embryos and improved IVF outcomes. These results are in agreement with previous studies, confirming the beneficial effect of PRP treatment on ovarian failure in women with diminished ovarian reserve. A case report of 19 women showed an increase in the number of retrieved oocytes after PRP treatment [21]. Moreover, two patients experienced spontaneous conceptions. In another prospective study (152 women), intraovarian injection of autologous PRP resulted in a significant decrease in basal FSH levels and an increase in the number of MII oocytes and cleavage stage embryos [22]. Similar results were reported by Pantos et al. [23] describing the effect of autologous PRP treatment on 3 patients. PRP treatment has been shown to have also a beneficial effect on follicular growth and maturation [24]. Other studies reported successful biochemical pregnancy in a prematurely menopausal woman after autologous PRP intraovarian infusion [11], and poor responders giving live births [12].
As the oocyte and embryo quality appears significantly different in both cycles (prior to and post PRP treatment), the mechanism for oocyte and embryo morphological improvement after PRP ovarian injection may be related to the wide field of action of the growth factors and cytokines contained in PRP. The communication between platelets and plasma proteins leads to fibrin clot formations that serve as a reservoir of these growth factors. They are then released then their release into the plasma occurs upon activation during tissue regeneration. These benefactor molecules include interleukin-8, platelet-derived angiogenic factor, platelet derived growth factor, insulin-like growth factor, stromal cell-derived factor 1, vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor and transforming growth factor beta [25], fibronectin, vitronectin and sphingosine-1-phosphate [4]. These PRP elements are a large group of regulatory proteins which can bind to cell membrane receptors and mediate essential signals [20]. unlike hormones, they show activity that is physiologically relevant in a very close proximity to their release site [26]. The observed short distant effects include chemotaxis, controlled release of different growth factors, angiogenesis, mitogenesis and formation of the extracellular matrix [27]. For instance, SDF-1 supports CD-34-positive progenitor cell recruitment and the process of target tissue remodeling [28], while PDGF guide cell migration [29]. Rescue from the developmental arrest also depends on PDGF [20], because this cytokine triggers DNA synthesis and entry into mitosis [30]. It was suggested that these specific PRP components might supply the key signals needed to induce precursor or stem cell differentiation into a mature oocyte [20].

Conclusions
Our study revealed that PRP treatment led to a significant increase in the number of the retrieved MII oocytes and the number of embryos successfully developed to day 5. Furthermore, we found that PRP ovarian treatment resulted in improved oocyte and embryo quality in approximately three-fourths of our patients. The application of this therapy would therefore likely increase the success rate of IVF cycles in poor responders. The treatment protocol utilized in this study could be routinely implemented in IVF clinics. With the continual ageing of the patient population seeking fertility treatment, the significance of such methods will also increase.