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Original Article

A novel method of intraovarian instillation of platelet rich plasma to improve reproductive outcome in young Indian women with diminished ovarian reserve

Parikh, Firuza R. MD, PhD; Sawkar, Sujatha G. MD; Agarwal, Sapna MD; Makwana, Prashant K. MSc; Khandeparkar, Meenal S. MS; Naik, Nandkishor J. MSc; Sanap, Mangesh V. MSc; Joshi, Spruha P. MSc; Athalye, Arundhati S. PhD

Author Information
Global Reproductive Health: Summer 2022 - Volume 7 - Issue 2 - p e59
doi: 10.1097/GRH.0000000000000059

Abstract

In the backdrop of advancing age at first conception, infertility has reached epidemic proportions across the world, including in India1. It is estimated that India is home to around 15–20 million couples with infertility. This represents nearly a quarter of the entire world’s estimated number2. At our center, we have observed a previously reported alarming trend where young women aged 35 years and younger are presenting with diminished ovarian reserve (DOR) and poor reproductive outcomes3,4. Another recent study from India has reported similar results5. Although there are many definitions for DOR and poor ovarian response (POR), these are used interchangeably6. It is acknowledged that the diagnosis of DOR has evolved from using clinical judgment to introducing ovarian markers like antral follicle count (AFC) measurements and estimation of anti-Müllerian hormone (AMH)7,8. We went by the accepted definition of DOR represented by a small number of antral follicles and low AMH in young women younger than 40 years with the presence of menstrual cycles. Our inclusion criteria were AFC<5–7 follicles, AMH levels of ≤1.5 ng/mL, and a prior history of poor fertility outcomes even with assisted reproductive technologies. Although traditionally, DOR is represented by AMH values of 1.1 ng/mL, we took a higher cutoff, as literature suggests that Indian women age 6 years earlier than Caucasian women9. This increasing trend of ovarian insufficiency is creating a generation of young Indian women with diminished reproductive potential10,11. For most of these young women, accepting oocyte donation is not an option due to ethical, social and religious reasons. Also since these women are young, many prefer to keep the option of donor eggs as a last resort. Thus, novel approaches to achieve a positive reproductive outcome are needed to alleviate their condition.

Ever since its introduction in 1954, platelet rich plasma (PRP) has seen a wide range of medical applications ranging from wound healing, reconstructive and plastic surgery, orthopedics, regenerative medicine and muscle healing12–14. Recent literature has propounded the beneficial effects of PRP in many reproductive conditions15. PRP has improved the reproductive outcomes in patients with refractory endometrium, repeated implantation failures, and endometritis due to genital tuberculosis16–18. Pregnancies have also been reported after instilling PRP in the ovaries of menopausal women19, as also in young women with DOR, POR, primary ovarian insufficiency, and poor responders20–22.

PRP is prepared from autologous blood samples of patients, and has 3–5 times the concentration of platelets than in circulating blood. PRP is also rich in cytokines and a large assortment of growth factors (GFs), including platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor (VEGF), growth differentiation factor 9, transforming growth factor-β, and various other GFs23. These GFs have mitogenic, chemotactic and neovascular properties, and are responsible for cell migration and differentiation, cell proliferation, activation of angiogenesis, and tissue regeneration24,25. In vitro and animal studies in rabbits, rats, and goats have also shown these beneficial effects25. In addition, it has been documented that the platelets in PRP attract stem cells to the site of injury, and these stem cells have known roles in cell regeneration and cell proliferation26.

These proliferative and cell-differentiating effects seen with the GFs and platelets contained in PRP are thought to be the underlying mechanism for the beneficial effects of PRP in reproductive medicine. It is thought that PRP treatment of ovaries enhances ovarian regeneration by promoting follicle formation, matrix and collagen formation, angiogenesis, cellular proliferation and differentiation, thereby having a potential to improve reproductive outcomes27–29.

Recent literature, although sporadic, has indicated that intraovarian instillation of PRP [intraovarian platelet-rich plasma (IOPRP)] may potentially have beneficial effects on the ovary. In the recent past, many more studies have emerged22,30, and it will be a matter of time before protocols are established in this field. At our center, we have started IOPRP instillation in young Indian women with DOR and the initial results have been encouraging31. There are technical difficulties in the procedure due to difficulty in accessing ovaries of such women and the intraovarian instillation of adequate volume of PRP. To overcome these difficulties, we have introduced a modified technique of IOPRP instillation. In the present study, we have evaluated the impact of our innovative technique of IOPRP instillation on the AFC, AMH levels, total and mature oocyte yield, and pregnancy outcomes. Our hypothesis was that IOPRP instillation by our technique would result in an improvement in AFC, and the growth promoting effects of PRP on the ovarian follicles would enhance the pregnancy outcome.

Methods

Study setting and ethical issues

This prospective, ongoing cohort study was performed at a tertiary care hospital. The study protocol was approved by the Institutional Scientific Advisory Committee and the Institutional Ethics Committee, and the study was carried out following the ethical principles of human research as enshrined in the 1964 Declaration of Helsinki and subsequent and relevant amendments. All participants provided voluntary written informed consent before their enrolment into the study.

Patients

A total of 116 women attending our fertility center with history of poor reproductive outcomes in the past and being diagnosed with DOR were screened for suitability for inclusion in the study. For this study, women with previous failed attempts at intrauterine insemination (IUI), women in whom in vitro fertilization (IVF) attempts did not result in ongoing pregnancy, and young women with unexpectedly low AMH and AFC were considered to have poor reproductive outcomes. The inclusion criteria were age below 39 years, menstruating, having a baseline AMH of ≤1.5 ng/mL, a baseline AFC of 5–7 follicles or less and having a history of infertility of ≥4 years. We selected a cutoff of AMH ≤1.5 ng/mL in our study, based on literature supporting the fact that Indian women age 6 years earlier than Caucasian women9. The exclusion criteria were women in established menopause, women with known genetic causes for DOR, women having secondary causes of infertility such as recurrent pelvic infections, previous ovarian surgery, history of receiving chemotherapy, endometriosis, and acute or chronic endometritis. Patients fitting the inclusion criteria were enrolled in the study, and those patients who were agreeable to participate in the study were asked to provide written voluntary informed consent before being recruited (group A, n=45). In order to establish the efficacy of this method, the pregnancy rates in 51 women with DOR with the same inclusion criteria and demographics but who had not undergone IOPRP during their IVF cycles during the same time period were compared (group B, n=51). This group of women did not undergo PRP due to their inability to go through 3 cycles and their desire to move on to the option of donor eggs if pregnancy did not occur with their own eggs. In group B, 13, 16, and 22 women had 1, 2, and 3 cycles of IVF, respectively.

Visits and data

The visit schedule and data collected during the visits in group A are summarized in Figure 1. Briefly, for all patients, 3 cycles of IOPRP instillation (IOPRP-1, IOPRP-2, IOPRP-3) were planned after the baseline visit. At the baseline visit, demographic data was recorded. AMH was recorded using serum enzyme chemiluminescence immunoassay (Roche Diagnostics), and AFC was recorded using transvaginal ultrasound done by the same operator. Subsequently, patients were initiated on the first mild ovarian stimulation (MOS-1) protocol which started from day 2 of the menstrual cycle (visit 2a). Baseline values of AMH and AFC were recorded 2 days before the expected period or on day 1 or day 2 of the menstrual cycle, just before starting the ovarian stimulation. The IOPRP-1 was done during the first egg retrieval (visit 2b) during which time the number and composition of eggs retrieved were recorded; since these values were representative of the pre-IOPRP status of the eggs, these corresponded to the baseline values of this outcome. The subsequent visits followed the same pattern, with visits 3a and 4a representing initiation of the MOS protocols prior to IOPRP-2 and IOPRP-3, respectively. During these visits, the values of AFC and AMH were collected. IOPRP-2 and IOPRP-3 were performed during visits 3b and 4b respectively. During each visit, patients were asked to report any adverse events. Patients who had medical, social, or economic reasons for discontinuing from the study after IOPRP-1 or IOPRP-2 were allowed to drop out of the study. Also, patients who conceived after IOPRP-1 or IOPRP-2 did not undergo IOPRP-2 and IOPRP-3, respectively. Some of the women with no oocytes, no embryos or abnormal embryos also chose to drop out prematurely with the idea of considering donor oocytes later.

F1
Figure 1:
Study flow, visits and data, and patient disposition. BL indicates baseline; MOS, minimal ovarian stimulation.

Technique of PRP preparation

For preparing PRP, we collected 60 mL of venous blood in sterile 10 mL EDTA collection tubes. The tubes were gently agitated. The first centrifugation was carried out at 1000 rpm for 10 minutes at room temperature to prevent platelet activation during centrifugation. After centrifugation, the blood separated into 3 layers. The supernatant layer along with the buffy coat (which contained the concentrated platelets) was collected in 3 tubes (Falcon #2095). This was further centrifuged at 3300 rpm for 5 minutes to form soft pellets of residual RBCs and platelets at the bottom of the tube. The supernatant platelet-poor plasma layer was collected in a tube (Falcon #2057) to be used as a buffer. The platelet pellet was resuspended in minimal platelet-poor plasma12,32. A total of 4 mL of PRP was prepared, with an intention of instilling about 1.5–2 mL of PRP in each ovary. The increased concentration of platelets in the final PRP suspension that was prepared just before the IOPRP instillation was validated in 10 PRP samples that we prepared and every sample was found to qualify33. Supplementary Table 1 (Supplemental Digital Content 1, https://links.lww.com/GRH/A1) shows the increased platelet concentration in these 10 samples.

Preparation for IOPRP instillation

Minimal ovarian stimulation (MOS): was performed with 100 mg of clomiphene citrate or 2.5 mg letrozole from day 2 to day 6 of the period; 150–225 units of hMG were added from day 3 till at least 1 follicle reached 17–18 mm in size. At this point, 10,000 IU of HCG was given as trigger; this was followed 34–36 hours later by follicular aspiration and IOPRP instillation. The mild ovarian stimulation facilitated in creating a space in which PRP could be instilled after follicular aspiration. For women with very low AMH, where folliculogenesis could not be completed and the follicle(s) remained static, the PRP instillation was performed when at least 1 follicle reached 14–15 mm in diameter.

Procedure of IOPRP instillation

The procedure was carried out using transvaginal ultrasound, under mild anesthesia. Usually each ovary would have 1–2 large follicles for instillation. All follicles more than 14 mm in diameter were aspirated with the objective of recovering oocytes. After aspirating the dominant follicle, the needle was kept in situ in the collapsed follicle and 1–1.5 mL of the PRP was gently instilled over 45 seconds. The refilling of the empty aspirated follicle with IOPRP was visualized through ultrasound. Then 1.0 mL of buffer (platelet-poor plasma) was instilled as a top-up to empty the tubing and needle of any PRP. No peritoneal leakage was observed on sonography. The next follicle was aspirated and the procedure was repeated. The follicular antrum was easily accessible in the minimally stimulated ovary. Another aspirated follicle was located and any remaining PRP was gently instilled. If any PRP solution remained, it was utilized by advancing the needle further into the ovarian stroma and the remaining 0.5 mL was instilled. The procedure was repeated in the other ovary.

Oocyte yield, fertilization, embryo culture, vitrification, and embryo transfer

The number of oocytes and the mature oocyte yield were recorded. ICSI was performed on all mature oocytes in every cycle. Embryos were cultured till blastocyst stage. As far as possible, we cryopreserved the embryos in both the groups at the blastocyst stage and performed blastocyst stage frozen embryo transfer unless the embryologist felt that cleavage stage embryos were unlikely to develop well. Serum bhCG was checked on day 14 of transfer. Women were followed-up for pregnancy and determined whether the pregnancy was biochemical or clinical. We did not evaluate fertilization and cleavage rates for the purpose of this study as these are dependent on variables like sperm parameters.

Outcomes

In the present study, we had 3 primary outcomes: changes in AFC and AMH levels and the changes in the number of total and mature oocytes after IOPRP. The secondary outcome of our study was establishment of pregnancies. The safety of the procedure was also recorded, and participants were asked to report on any adverse events observed in between the IOPRP cycles. In group B, the outcome of interest was the establishment of pregnancy. The number of oocytes retrieved during the first cycle in group B was higher (mean=5.00±3.24) than in group A (mean=3.11±2.54). This is because the stimulation for group B was as per established standard protocols and was not mild stimulation as in group A.

Statistics and data availability

All data was entered electronically and analyzed using Microsoft Excel 2016 and SPSS version 20. Paired t test was used to assess statistical significance in the changes seen with the AFC and AMH values collected at various points during the course of the study. The χ2 test was used to assess the significance of the pregnancy rates. A P-value of ≤0.05 was considered statistically significant for all tests performed.

Results

Patient disposition

Of the 116 women screened, 45 women were found to fit the inclusion criteria and were enrolled in the study. While all 45 women attended the first IOPRP instillation cycle, 33 women undertook the second cycle, and 19 women underwent all three cycles of IOPRP instillation. Twelve women could not complete 2 cycles and 14 could not complete 3 cycles due to medical, social, economic reasons and because 2 women reported natural pregnancy after IOPRP-1 and 2 women reported natural pregnancy after IOPRP-2 (Fig. 1). Some of the women who did not produce eggs or embryos or produced aneuploid embryos dropped out and a few opted for donor eggs.

Demographics

In group A, the average age was 34±2.8 years (range 26–39 y). Primary infertility was observed in 33/45 patients; the remaining 12 patients had secondary infertility. The average duration of infertility was 6.10±2.20 years (range 4–11 y). Mean AMH in group A was 0.92±0.41 ng/mL (range 0.18–1.5 ng/mL, n= 45). Previous history of having unsuccessfully undergone IUI and ICSI/IVF was recorded in 29 and 24 patients, respectively; 18 patients underwent both the procedures in the past. One patient had undergone as many as 7 IUI cycles in the past, and another patient had a history of 7 ICSI/IVF cycles. Previous natural conception and conception following IVF/IUI were documented in 5 and 4 patients, respectively; however, none of these conceptions resulted in ongoing pregnancy. In group B, the mean age was 33.60±3.09 years (range 24–39 y). The average period of infertility was 7.25±2.79 (range 4–14). Mean AMH was 0.98±0.38 ng/mL (range 0.26–1.5 ng/mL). In group B, 13, 16 and 22 women had 1, 2, and 3 cycles of IVF, respectively.

Primary outcomes

AFC

The average AFC value showed an increasing trend. The mean AFC values increased from baseline (3.44±2.35, n=45) through IOPRP-1 (3.89±2.21, n=45) and IOPRP-2 (4.91±2.79, n=33), and IOPRP-3 (4.95±2.84, n=19). Comparing AFC values between the sets of patients who underwent repeated IOPRP cycles, revealed that the AFC values consistently increased significantly with incremental IOPRP cycles, as signified by the mean difference between the average AFC values (Table 1). Compared with baseline values, the average AFC values were nonsignificantly higher after IOPRP-1 (n=45, mean difference 0.46, P=0.1198 vs. baseline at P<0.05) but significantly higher after IOPRP-2 (n=33, mean difference 1.27, P=0.0056 vs. baseline at P<0.05) and IOPRP-3 (n=19, mean difference 2.26, P=0.0002 vs. baseline at P<0.05).

Table 1 - Mean±SD AFC values at different study points.
AFC-1 (Before IOPRP-2) N=45 AFC-2 (Before IOPRP-3) N=33 AFC-3 (1-Month After IOPRP-3) N=19
Baseline AFC (before IOPRP-1) Common N=45 Common N=33 Common N=19
 AFC-BL: 3.44±2.35  AFC-BL: 3.64±2.52  AFC-BL: 2.68±1.63
 AFC-1: 3.89±2.21  AFC-2: 4.91±2.79  AFC-3: 4.95±2.84
Mean difference: 0.46 Mean difference: 1.27 Mean difference: 2.26
P=0.1198 (not significant at P<0.05) P=0.0056 (significant at P<0.05) P=0.0002 (significant at P<0.05)
AFC-1 (before IOPRP-2) Common N=33 Common N=19
 AFC-1: 3.82±2.42  AFC-1: 3.11±2.21
 AFC-2: 4.91±2.79  AFC-3: 4.95±2.84
Mean difference: 1.09 Mean difference: 1.84
P=0.0073 (significant at P<0.05) P=0.0011665 (significant at P<0.05)
AFC-2 (before IOPRP-3) Common N=19
 AFC-2: 4.11±2.13
 AFC-3: 4.95±2.84
Mean difference: 0.84
P=0.05387 (not significant at P<0.05)
P-value through paired t test.
AFC indicates antral follicle count; BL, baseline; IOPRP, intraovarian platelet-rich plasma.

AMH

Baseline AMH values were available for all 45 women; however, post-IOPRP-1 and post-IOPRP-2, AMH values were available for 26 women. AMH values were not collected after IOPRP-3 for several reasons including the added cost, some women considered the last IOPRP instillation as the end of the process, some dropped out after the second IORP installation and some conceived. The average values of AMH at baseline, post IOPRP-1 and post IOPRP-2 were 0.85±0.44 ng/mL, 0.85±0.47 ng/mL, and 0.94±0.53 ng/mL, respectively. The changes in average AMH levels after IOPRP-1 were not statistically significant when compared with baseline AMH values (P=0.4221 vs. baseline at P<0.05) but showed significance after IOPRP-2 (P=0.0482 vs. baseline at P<0.05).

Total number of oocytes and mature oocytes retrieved post the first and second IOPRP cycles

Complete data was available for 33 and 19 women who had undergone IOPRP-1 and IOPRP-2, respectively. The increase in the number and maturity of oocytes retrieved post-IOPRP-1 was not significant. However, there was a significant increase in both the number (P=0.0397<0.05, n=19) and maturity of oocytes (P=0.0125<0.05, n=19) following IOPRP-2 (Table 2).

Table 2 - No. of total and mature oocytes retrieved at the time of second and third IOPRP instillation.
Average Number of Total Oocytes Retrieved at Baseline Average Number of Total Oocytes Retrieved at IOPRP-2 Average Number of Total Oocytes Retrieved at IOPRP-3
n=19 n=19 n=19
47/19=2.47±2.44 61/19=3.21±3.36 77/19=4.05±3.54
P=0.0397 (significant at P<0.05)
n=33 n=33 NA
102/33=3.09±2.77 117/33=3.55±3.11 P=0.2577 (not significant at P<0.05)
No. mature (MII) oocytes retrieved at Baseline No. mature (MII) oocytes retrieved at IOPRP-2 No. mature (MII) oocytes retrieved at IOPRP-3
n=19 n=19 n=19
31/47=66% 48/61=78.7% 62/77=80.5%
P=0.0125 (P significant at <0.05)
n=33 n=33 NA
72/102=70.6% 92/117=78.6%
P=0.1373 (not significant at P<0.05)
The total number of oocytes and mature oocytes retrieved were significantly higher than baseline after 2 IOPRP (N=19 at P<0.05).
However there was no significant increase in the total number of oocytes and mature oocytes retrieved after 1 IOPRP (n=33 P>0.05).
IOPRP indicates intraovarian platelet-rich plasma; NA, not applicable.

Secondary outcome

Pregnancies

Frozen embryo transfer (FET) was done in 32/45 women. The data of the remaining 13 women is as follows; 8 women who had undergone IOPRP did not reach the stage of embryo transfer (as 1 did not produce eggs, 6 did not produce embryos, and 1 produced aneuploid embryos.) Natural conception occurred in 5 women. In all, 16/32 women who underwent embryo transfer conceived. Of these, 1 was a biochemical pregnancy and 15 clinical pregnancies were established (clinical pregnancy rate 46.88% per embryo transfer). Hence, totally, there were 20 clinical pregnancies in 45 women (overall clinical pregnancy rate was 44.45%). Four of 15 women (26.6%) had a miscarriage. In group B, 44/51 women underwent FET, 12 pregnancies were established. Of these 1 was a biochemical pregnancy, hence 11 clinical pregnancies were established (clinical pregnancy rate 25% per embryo transfer). Of the remaining 7 women who did not have FET, 1 conceived naturally (but it was a biochemical pregnancy) and 6 did not have embryo transfer as no embryos were formed. Hence there were a total of 11 clinical pregnancies in 51 women (overall clinical pregnancy rate was 21.57%). Miscarriage occurred in 6/11 women (54.55%). The overall clinical pregnancy rate in group A was significantly higher being 44.45% as compared with 21.57% in group B (P=0.0009<0.05). The miscarriage rate was higher in group B (54.55%) as compared with group A (26.6%).

Safety

Over the course of this study, no new safety signals of concern were observed. The overall incidence of adverse events following IOPRP instillation was as per reported literature and included mild pelvic pain which lasted for 24 hours in 9 women. No serious adverse events requiring hospitalization or treatment discontinuation were reported. No medical reasons for discontinuing IOPRP instillation and ICSI cycles were observed, except discontinuation of the subsequent IOPRP cycle in case of conception.

Discussion

The main findings of our study are that IOPRP instillation in mildly stimulated ovaries resulted in a statistically significant increase in the AFC while not having a consistent effect on AMH. There were a total of 20 clinical pregnancies in 45 women (clinical pregnancy rate 44.45%). This gains significance because all the 45 women included in our study had a poor reproductive history and were diagnosed with DOR, which meant that they had compromised reproductive potential. Twenty-four of these women had been offered the option of donor eggs at other clinics and initially all had rejected that option. Even though DOR is often described as the woman having an AMH of <1.1 ng/mL, we chose a higher value of ≤1.5 ng/mL in our study, since we have observed in our center that the AMH values among women with infertility are slightly lower when compared with the published literature which is largely based on values of Caucasian women3,4. Another study also reported similar findings5. This is further supported by a study by Iglesias and colleagues in which the ovarian reserve markers, AMH and AFC were found to have ethnic variations. Although the average age of Indian women undergoing their first or second IVF cycle was significantly less than Spanish women, they had similar ovarian reserve markers (AMH and AFC); this suggested that there was a 6-year advancement in ovarian aging in Indian women9. This prompted us to select a higher AMH cut-off of ≤1.5 than what is described in literature for DOR.

Unstimulated ovaries have a small volume and are dense; because of this, performing IOPRP by injecting an amount of 2.5–3 mL of PRP into such ovaries, under transvaginal ultrasound guidance, is technically difficult. Previous reports have documented peritoneal spills when 4 mL of PRP was instilled into the ovary34. Also, the technical feasibility of instilling 4mL of PRP in unstimulated ovaries has been questioned35. We bypassed the difficulty of access to the ovarian tissue by carrying out MOS with clomiphene citrate or letrozole, hMG, and HCG, as described earlier. By this method, there was an increase in the volume of the ovaries and we were able to successfully instil up to 4 mL of PRP into the ovaries, without any peritoneal spillage.

Performing IOPRP in mildly stimulated ovaries was also aimed at taking advantage of the increased vascularization and angiogenesis that is normally observed in the periovulatory period. VEGF is an important angiogenesis molecule. Since the ovulatory follicle and the subsequent corpus luteum have proliferation of blood vessels, there is a possibility that the VEGF present in the instilled PRP would have brought about vascular activation and neoangiogenesis in the ovaries, thereby contributing to some of the beneficial outcomes observed in our study36. The mediators present in PRP regulate angiogenesis and tissue perfusion either awakening the latent oocytes or establishing communication with uncommitted ovarian stem cells to differentiate and develop into de novo oocytes20. GFs like BMP-2, BMP-4, and GDF-5 which are present in PRP also play an important role in the development of mesenchymal and progenitor stem cells37. These features may play an important role in improving oocyte competency and thereby enhancing pregnancy rates. GFs like VEGF and bFGF are mitogenic and contribute to vascularizing the granulosa, giving functionality to the early corpus luteum38. It has been proposed that the ovarian epithelium experiences injury at the time of follicular rupture and the surface epithelium undergoes repetitive damage. The stem cells in this area help with healing26. These observations suggest that IOPRP instillation may cause injury to the ovarian surface allowing a similar process thereby enhancing the ovarian milieu.

Preantral follicles take 85–90 days to mature; also, around 85 days are required for the growth of the secondary oocyte (class 1) to the preovulatory oocyte (class 8)39. Given this background, we decided to evaluate the effect of IOPRP over 3 minimally stimulated cycles which approximately span over 90 days. In our study, the AFC values after 3 cycles were significantly higher than baseline AFC values. These findings were similar to the findings of Melo et al who reported a statistically significant increase in AFC in 46 women who underwent 3 cycles of IOPRP instillation from a median of 4 follicles (interquartile range—3–5) pretreatment to a median of 7 follicles (interquartile range—6–8) post-IOPRP (P<0.001)40. They also used a similar rationale for advocating 3 IOPRP instillation cycles.

Although we proposed to the women that the cycles should be done successively, not all women could comply. In some women, the next cycle could not be started due to residual cysts from the previous cycle or due to various medical and social reasons.

AMH is secreted by primary preantral and small antral follicles and does not show variation throughout the menstrual cycle. A decline in AMH is often considered to be the earliest marker of decline in ovarian reserve8. In our study, serum for AMH was not collected from all patients, and we did not collect serum for AMH values after IOPRP-3; this might have contributed to the inconsistent AMH levels in our study. The average values of AMH at baseline, post-IOPRP-1 and post-IOPRP-2 were 0.85±0.44 ng/mL, 0.85±0.47 ng/mL, and 0.94±0.53 ng/mL, respectively. The changes in average AMH levels after IOPRP-1 were not statistically significant when compared with baseline AMH values (P=0.4221) but showed significance after IOPRP-2 (P=0.0482 at P<0.05). Scott Sills et al20, also remarked that in their study, although there was a rise in AMH after IOPRP injection, it was insignificant.

The contribution of racial differences to the effect of IOPRP instillation on AMH levels cannot be ruled out as well. Further studies involving larger sample size, with more consistent data collection are required to verify the findings of our study regarding AMH. Having acknowledged this shortcoming in AMH assessment we also want to stress that the resurgence of ovarian activity following PRP is unlikely to do with increased recruitment of primordial follicles as it would have taken more than 90 days for these follicles to become hormone sensitive39. It is more likely that PRP stimulates the development of pre-existing follicles or prevents their atresia40, indicating that AMH may not be a suitable indicator for determining the utility of PRP instillation. Perhaps that could address the issue of why we did not see much improvement in AMH in these women after the IOPRP instillations. The occurrence of several natural conceptions after the IOPRP instillation also points to the activation of preexisting follicles.

One of the ambiguities of treatment for enhancement of ovarian response arises due to the disparity in the nomenclature used to describe failing ovarian function. There are 2 distinct groups of women being treated, resulting in inconsistency in reporting by various researchers. One group consists of women who are menopausal with almost nondetectable AMH. Sporadic success has been reported with the use of PRP in menopausal women. The other group consists of women below the age of 40, with presence of menstrual cycles, with DOR, and low AMH values. Our study was specifically targeted toward women younger than 40 years, with a poor ovarian reserve, an AMH ≤1.5 ng/mL, with infertility of ≥4 years and/or repeated IVF failures and no live births. Our primary aim was to explore the extent to which IOPRP instillation was able to enhance ovarian response in young women with this condition and to augment their chances of conception. We did not extend this study to older and menopausal women as there are shortcomings in treating such women due to increased genetic abnormalities in the oocytes.

Our study is not without limitations. It can be argued that the conceptions following IOPRP could be by chance. However, the significant improvement in AFC, and the establishment of successful IVF and natural pregnancies in these women who had a prolonged history of infertility with failed IVF cycles in the past and who were suggested to opt for donor eggs, is a pointer that IOPRP has a beneficial role for these women. Also the pregnancy rates were significantly enhanced in group A as compared with group B. Finally, given the small sample size of this study, a well-designed prospective clinical trial with a larger sample size, more rigid inclusion and exclusion criteria, and a more robust statistical analysis would be needed to further validate the reproductive benefits of IOPRP instillation that we have alluded to in our study.

Conclusions and way ahead

This is an ongoing study which has demonstrated the potential of IOPRP instillation in improving reproductive outcomes in women who have exhausted standard methods of treatment. IOPRP is a simple, minimally invasive, and less expensive procedure, compared with laparoscopic IOPRP instillation. MOS is also cost-effective compared with conventional ovarian stimulation which would require a considerably higher dose of gonadotrophins; obtaining an increased number of eggs after minimal stimulation is therefore cost-effective. In our study, we were able to establish 20 clinical pregnancies among 45 young women with diminished reproductive potential, following IOPRP. These women were not agreeable to oocyte donation as an initial option; from our viewpoint, this is an indicator that IOPRP might be clinically efficacious. IOPRP instillation can be repeated as required, without any morbidity as we did not observe any detrimental effects after repeated instillation of PRP. It does not require an extensive setup and can be combined with follicular aspiration. Our study indicates that IOPRP can significantly improve AFC, without significantly altering AMH levels. More importantly, among women who are poor responders to assisted reproductive technologies, IOPRP appears to be efficacious in establishing clinical pregnancies. Our study may open up future research avenues to unravel some of the pathways explaining the efficacy of PRP in ovarian rejuvenation.

Conflicts of interest disclosure

The authors declare that they have no financial conflict of interest with regard to the content of this report.

Acknowledgments

The authors wish to acknowledge the contribution of all members of the Department of Assisted Reproduction and Genetics, Jaslok Hospital and Research Centre, Mumbai.

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Keywords:

IOPRP; Ovarian rejuvination; Intraovarian platelet-rich plasma

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