Study of role of PRP (platelet rich plasma) in thin endometrium in patients with infertility: an open label randomized controlled study

INTRODUCTION

According to the World Health Organisation, incidence of infertility is about 10% worldwide.  According to a recent report globally, 48.5 million couples are affected by infertility. This report states that that nearly every 7th couple is infertile[1]. In India, the prevalence of primary infertility has been reported to be in the range of 3.9 to 16.8%[2]. The term infertility is used when “a couple fails to conceive after one year of regular sexual activity without contraception”. WHO defines this condition in following words, “a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse”[3]. Eighty per cent of all women desiring children, conceive within 1 year of married life while another 10% within the second year. Another 10-12% of all the other couples have only one child and wish to have more[4].

The reasons for infertility could be multiple. Both males as well as females could be responsible for it. Identifiable factors affecting female infertility include: “hormonal or endocrine disturbances (menstruation or ovulation problems), tubal pathologies (occlusions, pelvic adhesions and other tubal abnormalities), non-tubal pathologies (cervical or uterine disturbances), sexual dysfunction and congenital abnormalities”[5].Among different causes of infertility, male factor besides being to sole cause of infertility in 20% of infertile couples may be contributing factor in as many as 40% of cases. Female factor contributes about 40% of the cases. Disorders of ovulation such as an ovulation account for about 20% to 40% of all cases of female infertility. Tubal factor such as tubal injury, blockage, or paratubal adhesions account for 30-40% of cases of female infertility. Cervical factor is estimated to be a cause of infertility in no more than 5% of infertile couples. Other factors include “decreased ovarian reserve, peritoneal factors, uterine factors, immunologic factors, systemic conditions (including infections or chronic diseases such as autoimmune conditions or renal failure), and unexplained factors (including endometriosis with no evidence of tubal or peritoneal adhesions)”.

Over the last few decades, in vitro fertilization (IVF) has gained immense popularity and has emerged as a ray of hope for the infertile couples. Today, in developed countries like the USA and Europe, IVF is responsible for 1.6% and 4.5% of total live births respectively[6]. However, even in vitro fertilization is not always successful. Almost 20 to 25% of suitable women fail to respond to IVF even after five cycles despite significant improvements in its success rate over the last decade[7].

Among various factors associated with a failed implantation, endometrium plays an important role. Endometrial thickness is one of the contributory factors in determining the successful implantation and pregnancy[8],[9]. A number of studies report endometrial thickness >7 mm as the optimum for successful embryo transfer10,[10],[11]. From the point of view of IVF, thin endometrium is defined as <7 mm on the day of ovulation, or on the day of human chorionic gonadotrophin (HCG) injection in fresh in vitro fertilization (IVF) cycles, or the day to start progesterone in frozen-thaw embryo transfer cycles[12],[13].

Therapies for thin endometrium include “long-term administration of estrogen, low-dose of aspirin, vitamin E supplement, vaginal sildenafil citrate application, electroacupuncture and transvaginal endometrial perfusion of granulocyte colony stimulating factor (G-CSF)”. Despite existence of so many treatment alternatives, a large proportion of women do not show response to these treatments. Thus, there is a need for other options for patients with thin endometrium13,[14],[15]. Estradiol valerate is one of the commonly used and established modalities for management of thin endometrium during IVF[16].

During the recent years, regenerative medicine has emerged as a process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish the normal function. This process is done through stem cell isolates, recombinant growth factor amplification and platelets augmentation, respectively[17],[18].

In recent years, a preparation called Platelet rich plasma (PRP) is emerging as a platelet augmentation technique. PRP is characterized by its natural autologous nature that has been shown to have a large potential in ensuring tissue regeneration. The growth stimulus of PRP comes from the alfa granules found in platelet that speed up the tissue recovery by acting as catalyst to the natural healing and tissue repair processes. They also induce the production of new collagen by “the fibroblasts, osteoblasts and chondrocytes as per need of the parent tissue”.

The scope of PRP use has been described in a host of medical fields like orthopedics, cosmetology, reconstructive surgery, dentistry, orthopedic surgery and sports medicine by harnessing the inherent property of platelets by facilitating secretion of various growth factors. PRP has also gained popularity as an alternative option for treatment of thin endometrium[19],[20]. Platelet-rich plasma (PRP) derived from fresh whole blood and has anti-inflammatory and pro-regenerative functions. After the activation of the platelets in PRP, “growth factors as vascular endothelial growth factor (VEGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are actively secreted and transformed into their bioactive forms within 10 minutes after clotting. These growth factors are known to regulate cell functions such as attachment, migration, proliferation, differentiation and promote extracellular matrix accumulation. Therefore, PRP has been widely used for various therapeutic areas, for example, promoting healing process in orthopedics, ophthalmology and healing therapies”[21],[22],[23].

Given the encouraging role of PRP in management of thin endometrium as evidenced in some of the recent studies, the present study is being planned to study the role of PRP (Platelet Rich Plasma) as compared to EV (Estradiol Valerate) in management of thin endometrium in patients with infertility using an open label randomized controlled trial.

AIM AND OBJECTIVES

AIM

To study the role of PRP (Platelet Rich Plasma) as compared to EV (Estradiol Valerate) in management of thin endometrium in patients with infertility.

OBJECTIVES

1.         To study and compare the effect of PRP and EV on endometrial thickness among infertile women undergoing IUI.

2.         To study and compare the effect of PRP and EV on successful implantation rates.

3.         To study and compare the effect of PRP and EV on successful chemical and clinical pregnancy rates.

4.         To study and compare the effect of PRP and EV in terms of adverse events like abortions.

 REVIEW OF LITERATURE

Zadehmodarreset al. (2017)[24] in a pilot study that included 10 women with thin endometrium (ET<7 mm) undergoing assisted reproductive technology treatment used PRP for management of PRP. Before PRP injections, endometrial thickness ranged from 4 to 6 mm. After 48 hours of first PRP injection, endometrial thickness ranged from 5.2 to 6.8 mm. The patients were given a second PRP injection. After 48 hours of second PRP injection, endometrial thickness reached >7 mm in all the cases. Successful implantation was achieved in all the cases. Successful chemical pregnancy was achieved in 50%. Successful clinical pregnancy rate was 40%. The findings of the study showed a promising role of PRP in management of thin endometrium and subsequent favourable clinical outcomes.

Eftekharet al. (2018)[25] in an RCT evaluated the effect of PRP injection on endometrial thickness and subsequently on successful implantation and clinical pregnancy rates as compared to controls. Mean increase in endometrial thickness within 48 hours of PRP injection was 2.58 mm as compared to 1.89 mm in the control group. Successful implantation was seen in 21% of PRP as compared to 9.37% of controls, thus showing a significant difference between the two groups. Per transfer and per cycle chemical, clinical and ongoing pregnancy rates were also higher in PRP as compared to the control group, but the difference between the two groups was found to be significant only for per cycle clinical pregnancy rates.  No significant difference between the two groups was observed for abortion rates.

Chang et al. (2019)[26]in their study that recruited 64 women with thin endometrium (<7 mm) undergoing frozen embryo transfer (FET) cycles randomized them to two groups. In the case group (n=34) intrauterine infusion of autologous PRP was done on the 10th  day and the day when progesterone was given in HRT cycle whereas women who did not receive PRP (n=30) were placed in control group. At the time of implantation, women receiving PRP had significantly thicker ET (7.65±0.22 mm) as compared to women in control group  (6.52±0.31 mm). Cycle cancellation rate was also lower in PRP group (19.05%) as compared to that in control group (41.18%). The implantation and clinical pregnancy rates were also found to be significantly higher in PRP as compared to that in control group (27.94% vs 11.67% and 44.12% vs 20%, respectively). The findings of the study highlighted the role of PRP in “promoting endometrium proliferation, improving embryo implantation rate and clinical pregnancy rate for women with thin endometrium in FET cycles”.

Kim et al. (2019)[27]in a prospective interventional study that included a total of 24 women who had a history of two or more failed IVF cycles. All thesewomen had refractory thin endometrium. They plannedintrauterine infusion of autologous PRP 2 or 3 times from menstrual cycle day 10 of their FET cycle as the intervention to manage thin ET.Assessment of ET was done 3 days after the final autologous PRP infusion. A total of 22 patients underwent FET. They found an average increase in ET to be 0.6 mm following PRP infusion. A total of 12.7% implantations were successful. Clinical pregnancy rate achieved was 30%. Ongoing pregnancy rate was 20% which was equal to live birth rate. There were 12 patients who showed an increase of ET by 1.3 mm following PRP infusion, however, they also noted a mean decline of 0.7 mm in ET of seven patients. There was one patient who did not show any change in ET following PRP infusion. No adverse effects were seen in any of the patients treated with autologous PRP. The authors found autologous PRP to be useful in restoring the endometrial receptivity of damaged endometrium in addition to increase in ET.

Kusumiet al. (2020)[28]investigated the effectiveness of intrauterine administration of platelet-rich plasma (PRP) in frozen embryo transfer (FET) cycle in 36 women having endometrial thickness <7 mm.  PRP was administrated on the 10thand 12thdays of the second HRT cycle. The authors observed a mean increase in endometrial thickness at day 14 was observed by 1.27 mm and 0.72 mm respectively on the basis of blinded and unblinded measurements. A total of 32 out of 36 (88.9%) women underwent FET. The authors noted a clinical pregnancy rate of 15.6%. No adverse event was encountered during the study. The authors found the PRP administration as an effective and safe procedure to increase the endometrial thickness and achieving clinical pregnancy.

Zargeret al. (2021)[29]evaluated the effect of intra-uterine infusion of platelet-rich plasma (PRP) in frozen embryo transfer cycles on endometrial thickness and clinical pregnancy rates among a total of 24 women having thin endometrium (<7 mm). Before PRP administration, mean ET was 6.15±0.38 mm which became 7.49±0.61 mm after PRP administration, thus showing a mean increase of 1.34 mm. All the women except two women achieved ET >7 mm. They recorded clinical pregnancy rate of 45.8%. Ongoing pregnancy rate was 37.5%. There was no ectopic pregnancy. The authors found PRP to be an effective therapy in management of thin endometrium.

Dogra et al. (2022)[30]reported a study in which a total of 20 women having endometrial thickness <7 mm undergoing IVF and FET cycles were subjected to intraueterine PRP administration. They received a total of 26 PRP cycles. Mean increase in endometrial thickness following first PRP administration was recorded as 1.07 and 0.83 mm respectively during fresh IVF and FET. PRP administration was helpful in increasing the endometrial thickness despite conditions like tuberculosis, low ovarian reserves and polycystic ovarian syndrome. Clinical pregnancy and live birth rates were 20% and 25% respectively. The authors did not note any adverse effect. On the basis of these findings, PRP was found to be  a safe and effective measure to increase the endometrial thickness and improving clinical pregnancy rates.

Desai et al. (2023)[31]in their study that included a total of 61 women aged between 22 and 40 years having endometrial thickness <7 mm even after receiving estradiol valerate therapy. There were 44 women having normal hysteroscopic findings while remaining 17 had abnormal hysteroscopic findings. In both the groups, prior to PRP administration, mean endometrial thickness was 6.49 and 6.00 mm respectively in the two groups. After 48 hours of PRP administration mean increase in endometrial thickness was 1.62 and 1.61 mm respectively and after 72 hours it was 2.42 mm and 3.28 mm respectively in the two groups. Clinical pregnancy rates were 50% and 52.9% respectively in women with normal and abnormal hysteroscopic findings. The implantation rate per embryo was 25.2% in normal and 26.8% in abnormal hysteroscopic groups. The findings of the study showed that PRP was helpful in increasing the endometrial thickness as well as in achieving good clinical pregnancy rates in women with or without hysteroscopic abnormalities.

Lacunae in Literature: Although usefulness of intrauterine PRP administration for management of thin endometrium has been enumerated in various studies, however, there are limited randomized controlled trials to assess its clinical efficacy in comparison with conventional management. Moreover, most of the emerging evidence is from abroad and there is lack of related studies from India. The present study intends to fill these gaps in literature.

 MATERIAL AND METHOD

Study Settings: Tertiary Care GCCU, Department of Obstetrics and Gynaecology, King George’s Medical University, Lucknow.

Study Population: Women undergoing infertility treatment at Department of Obstetrics and Gynaecology, King George’s Medical University, Lucknow.

Study Design: Randomized-Controlled trial.

Sample Size: In a previous study25, mean increase in endometrial thickness 48 hours after PRP injection was found to be 2.58 mm in PRP as compared to 1.89 mm in control group, thus showing a mean difference of 0.69 mm between the two groups. In the present study, we also expect a similar difference. The sample size was calculated using the following formula (Charan and Biswas, 2013)[32]:

N>2´(Zα/2+Zβ)2´s2/d2+ 10% Contignency

where n: Sample size per group

s: Pooled standard deviation = 1.00 mm (projected)

d: Difference in the means (effect size) = 0.69 mm (Ref. 25)

zα/2: Constant at 95% confidence level its value is 1.96

Zβ : Constant at 80% Power of the study its value is 0.84

Contigency multiplier = 1.10

n = 2´(1.96+0.84)2´12/0.682  x 1.10

= 2 x 7.84´1/0.4624´1.10

=37.3~37

Thus the calculated sample size is 37. However, after rounding it off to nearest even number to ensure equal randomization in two groups we will enrol a total of 38 patients and will randomize them into two groups of 19 patients each.

Sampling Frame

Inclusion Criteria

·        Women attending the infertility clinic and scheduled to undergo frozen emnryo transfer (FET).

·        Aged <40 years.

·        Endometrial thickness <7 mm

·        Basal FSH <10 IU/L

·        At least 2 frozen good-quality blastocyst-stage embryos

Exclusion Criteria

·        Abnormal findings on diagnostic hysteroscopy

·        Hematological disorders

·        Chromosomal or genetic abnormalities

·        Hormonal disorders

Permissions and Approvals

Approval for study will be obtained from the Institutional Ethics Committee. Informed consent will be obtained from all the patients.

Method

A total of 38 women falling in sampling frame will be enrolled in the study. At enrolment, sociodemographic details regarding age, place of residence (rural/urban), occupation, education, family income and type of housing will be noted.

Medical history and exposure to risk factors like tobacco chewing, smoking, alcohol use will also be noted.

Obstetric history, type of infertility, menstrual history and number of previous IVF attempts will also be enquired and noted.

All the patients will be treated using the standard HRT protocol, the endometrial priming will be started on the day 2 to 3 of each patient’s menstrual cycle. The patients will be then randomized into the following two groups:

Group A (n=19; experimental group): Patients who will receive intrauterine infusion of autologous PRP on the 10th day of cycle in addition to routine estradiol valerate therapy.

Group B (n=19; control group): Patients not receiving PRP and remaining on routine estradiol valerate therapy only.

Method of Randomization: Sequentially Numbered Opaque Sealed Envelope (SNOSE) method.

All the patients will receive estradiolvalerate orally administered with a start dose of 6 mg/d. The dose of estradiolvalerate will be increased according to the endometrium responseupto a maximum dose of 12 mg/d.

In the experimental group, PRP will be prepared from the peripheral blood drawn from the forearm. A total of 20 mL of blood will be collected in two 10 ml vacuum blood collection tubes. After centrifugation of blood at 2000g for a period of 6 minutes, a total of 1 mL of PRP will be obtained (0.5 mL from each tube). On day 10 of the cycle, the PRP collected by the above method will be infused into the uterine cavity using ET catheter under sonographic guidance.

At day 10, sonographic measurement of endometrial thickness will be done in both the groups.

The standard HRT protocol will continue unabatedly in both the groups.

A repeat sonographic assessment for endometrial thickness will be done in both the groups 48 hours after the PRP infusion / first endometrial thickness assessment.

Subsequently, “two good-quality blastocyst-stage embryos will be transferred to all patients. The criterion for embryo quality will be consulted from the embryo morphology assessment by the Istanbul consensus workshop, blastocysts scored as Grade 3 or 4 were considered as good-quality.  After embryo transplantation, the luteal phase will be supported by daily intramuscular injection of 40 mg progesterone, combined with 200 mg vaginal progesterone soft capsules every night. Serum human chorionic gonadotropin (hCG) level will be tested 14 days after embryo transfer. Vaginal ultrasonography will be performed 35 days after transfer in cases of a biochemical pregnancy, and a clinical pregnancy will be defined as the presence of an intrauterine fetalheart beat. Implantation rate will be determined by the number of gestational sacs over the total number of embryos transferred”.

The process will be repeated upto a maximum of two cycles.

The primary outcome measure for the study will be increase in endometrial thickness 48 hours after PRP administration. The secondary outcome measureswill be successful implantation, chemical and clinical pregnancy. Adverse events if any will also be noted.

Data Analysis

Data will be analyzed IBM SPSS Stas 21.0 or above software. Chi-square test, paired ‘t’-test and Independent Samples ‘t’-test will be used to compare the data. Confidence level of the study will be kept at 95%.

 WORKING PROFORMA

Name                                      Age                                         Residence: Rural/Urban

Occupation                            Education                              Family Income

Type of housing: Kutcha/Pucca                                         Socioeconomic Status

(Kuppuswamy, 2023)

Weight                                   Age                             BMI

Medical History

Tobacco                                             Smoking                                 Alcohol

Gravida                                              Parity

Menstrual History

Age at menarche                               Menstrual regularity: Yes/No

Marital History:

Time since marriage

Type of infertility: Primary/Secondary

Number of previous IVF Attempts

Medical History

 Endocrine disorder

Randomized Treatment group:      Experimental / Control

Day 10 Endometrial Thickness

Day 12 Endometrial Thickness

Number of embryos

Total

Grade 3 / 4

Type of embryo

Fresh / Frozen

IU Implantation: Successful / Failed

Chemical Pregnancy: Yes / No

Clinical Pregnancy: Yes / No

Adverse events

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