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A comparative evaluation of subendometrial and intrauterine platelet-rich

plasma treatment for women with recurrent implantation failure

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DOI: 10.1016/j.xfss.2021.03.002

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ORIGINAL ARTICLE: REPRODUCTIVE DISEASES

A comparative evaluation of
subendometrial and intrauterine
platelet-rich plasma treatment for
women with recurrent
implantation failure
Majiyd Abdul Noushin, M.D., M.R.C.P.I., F.R.M.,a Mohamed Ashraf, M.D., D.G.O., D.P.S.,a
Chaitra Thunga, M.D., D.N.B., F.R.M.,a Sankalp Singh, M.D., D.N.B.,a Swati Singh, M.D., D.N.B.,a
Reema Basheer, M.D., F.R.M.,a Raiza Ashraf, M.B.B.S., M.Sc., F.C.E.,a and Kanna Jayaprakasan, M.D., Ph.D.b
a
CRAFT Hospital & Research Centre, Kerala, India, and b Royal Derby Hospital, Derby, United Kingdom

Objective: To compare the effectiveness of treatment with autologous activated platelet-rich plasma (PRP), administered to either the
subendometrium (SE-PRP) or endometrial surface (intrauterine; IU-PRP), against controls.
Design: Prospective observational cohort study.
Setting: Tertiary fertility unit.
Patients: Women aged <40 years with a history of recurrent implantation failure undergoing frozen embryo transfer (FET) (n ¼ 318).
Interventions: In SE-PRP, PRP was injected into the subendometrial space transvaginally in the luteal phase of the previous cycle of
embryo transfer under ultrasound guidance (n ¼ 55). In IU-PRP, PRP was administered during the index FET cycle when the
endometrium was approximately 7 mm (n ¼ 109). Both SE-PRP and IU-PRP groups were administered 300 mg of granulocyte
colony-stimulating factor (G-CSF) subcutaneously once a day for 3 days to boost white blood cells (WBC) and growth factor
production in the PRP sample. The control group consisted of women who did not choose PRP treatment and underwent standard
FET with no intervention (n ¼ 154).
Main Outcome Measures: Ongoing pregnancy rate or live birth rate (OPR/LBR) per transfer cycle, clinical pregnancy rate (CPR) per
transfer cycle, and miscarriage rate.
Results: As a result, OPR/LBR was higher in the SE-PRP (22/55, 40%) and IU-PRP (45/109, 41.3%) groups than that in the control group
(34/154, 22.1%). It was similar between the SE-PRP and IU-PRP groups. Moreover, CPR showed a similar trend with a higher rate in the
SE-PRP (28/55, 51%) and IU-PRP (57/109, 52.3%) groups than that in the controls (52/154, 33.8%). No statistical difference in the CPR
was noted between the SE-PRP and IU-PRP groups. The miscarriage rate was similar in all three groups (14/55, 25.45%; 25/109, 22.23%;
and 34/154, 22.07%, respectively).
Conclusion: In women with a history of recurrent implantation failure, PRP treatment appears to improve FET outcome with an in-
crease in OPR/LBR. However, SE-PRP treatment does not offer any advantage over lesser invasive IU-PRP treatment. (Fertil Steril
SciÒ 2021;-:-–-. Ó2021 by American Society for Reproductive Medicine.)
Key Words: Intrauterine PRP, PRP, RIF, subendometrial PRP

Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfss-d-20-00082

Received December 7, 2020; revised March 2, 2021; accepted March 16, 2021.
M.A.N. has nothing to disclose. M.A. has nothing to disclose. C.T. has nothing to disclose. S.S. has nothing to disclose. S.S. has nothing to disclose. R.B. has
nothing to disclose. R.A. has nothing to disclose. K.J. has nothing to disclose.
Funded by a private tertiary level clinic and a couple who paid the cost of the treatment.
Reprint requests: Majiyd Abdul Noushin, M.D., M.R.C.P.I., F.R.M., CRAFT Hospital & Research Centre, Thrissur, Kerala 680664, India (E-mail: drnoshinashraf@
gmail.com).

Fertil Steril Sci® Vol. -, No. -, - 2021 2666-335X/$36.00

© 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.xfss.2021.03.002

VOL. - NO. - / - 2021 1

ORIGINAL ARTICLE: REPRODUCTIVE DISEASES

R
ecurrent implantation failure (RIF) is a relatively com- been reported on this concept. This study aimed to compare
mon problem with an overall reported incidence of the effectiveness of treatment with autologous activated
10% among patients who underwent assisted repro- PRP, administered to either the subendometrium (SE-PRP)
ductive technology (1). Although there is no accepted univer- or endometrial surface (IU-PRP), against controls.
sal definition for RIF, the commonly restated definition as per
Coughlan et al. (2) is the failure to achieve a clinical preg- MATERIALS AND METHODS
nancy after transfer of at least four good-quality embryos
This prospective observational cohort study was undertaken
in a minimum of three fresh or frozen cycles in a woman
in a tertiary fertility unit from March 2019 to May 2020 after
aged <40 years. Recurrent implantation failure could be a
securing approval from the Institutional Ethics Committee.
consequence of uterine, gamete- or embryo-related, and
Women aged <40 years with a history of RIF undergoing
immunologic factors or suboptimal laboratory conditions.
frozen embryo transfer (FET) (n ¼ 318) were included in the
After excluding embryo-related causes, poor endometrial
study. Recurrent implantation failure was defined as failure
receptivity is a major limiting factor for success in assisted
to achieve a clinical pregnancy after R3 embryo transfer
reproductive technology (3). Various treatment regimens
(ET) cycles with at least a total of four good-quality cleav-
including endometrial receptivity array, endometrial scratch-
age-/blastocyst-stage embryos derived from autologous gam-
ing, sequential transfer, immune modulators, and growth fac-
etes transferred in an endometrium of R8-mm thickness (2).
tors are considered to combat this issue (4–8). However, there
The exclusion criteria included women with a body mass
is little consensus on the efficacy of the treatment mentioned
index R 30 kg/m2, congenital and untreated acquired uterine
earlier (9).
abnormalities, untreated hydrosalpinges, poor ovarian
Platelet-rich plasma (PRP), a concentrate of autologous
responder as per the Bologna criteria, thrombophilia, or un-
PRP protein derived from whole blood, has been tried by several
controlled endocrine or hematologic dysfunction; those un-
clinicians to improve embryo implantation (10). Chang et al.
dergoing preimplantation genetic testing cycles; and those
(11) first described intrauterine infusion of PRP in patients
who had thin endometrium (<8 mm) in the index FET cycle.
with thin endometrium. Subsequently, a few additional studies
Severe male factor infertility, difficult ET, only poor-quality
have also suggested the role of PRP in patients with refractory
embryos available, and couple with genetic and chromosomal
thin endometrium (12). Reversal of red blood cell (RBC) to
abnormalities were also excluded from the study.
platelet ratio by decreasing RBC to 5% (which is less beneficial
All patients underwent antagonist protocol for controlled
for healing process) and concentrating the platelets up to 94%
ovarian stimulation as per routine hospital protocol. All pa-
(which, when activated, releases growth factors and cytokines)
tients with RIF underwent ovarian reserve testing, basal hor-
is the principle behind the use of PRP (13). Recently, intrauter-
monal evaluation, karyotype evaluation, and acquired
ine infusion of PRP was evaluated and utilized for endometrial
thrombophilia testing. Routine ultrasonography (USG) was
growth and receptivity in several studies including a meta-
also a part of the evaluation, and if there was any suspicious
analysis showing positive benefit, while a few studies did not
2-dimensional USG finding, then 3-dimensional USG and
find a conclusive beneficial outcome (11–12, 14–18). This
confirmatory laparoscopy and or hysteroscopy were per-
discrepancy was mainly due to the non-standardization of
formed. Their male partners were also evaluated, and neces-
PRP preparation and the technique of administration.
sary treatment measures were taken.
Platelet-rich plasma through its paracrine action recruits a
After fulfilling the inclusion and exclusion criteria, study
wide array of cytokines and growth factors like transforming
participants were selected (Fig. 1). The proposed treatment
growth factor (TGF)-b, platelet-derived growth factor (PDGF),
with instructions, benefits, and side effects were explained
insulin-like growth factor-1, vascular endothelial growth fac-
to all patients with RIF with patient information leaflets.
tor, epidermal growth factor, hepatocyte growth factor, and
The patients were given the option of SE-PRP, IU-PRP, or
interleukin (IL)-8 (19). Similarly, the activation of PRP aug-
no intervention, and their choices were documented and in-
ments the production of growth factors (PDGF-AA, PDGF-
terventions were assigned as per the following.
AB, PDGF-BB, TGF-b1, TGF-b2, and epidermal growth factor),
anti-inflammatory cytokines (IL-4, IL-13, and interferon-a),
and pro-inflammatory cytokines (IL-8, IL-17, and tumor necro- Interventions
sis factor-a). These components are known to increase endo- Participants who chose subendometrial (SE-PRP group,
metrial receptivity and endometrial microvasculature (20). N ¼ 55) were administered subendometrial injection of autol-
Since PRP is autologous and is derived from one’s own blood, ogous activated PRP in the luteal phase of the previous cycle
there is no risk of viral infections and immunologic reactions, of ET between cycle days 21 and 24. Participants who chose
and it is considered safe compared with other interventions intrauterine PRP (IU-PRP group, N ¼ 109) were administered
in RIF (11, 21). autologous intrauterine activated PRP during the index FET
We introduced a novel technique of administration of preparation cycle. Participants who did not choose for PRP
activated PRP into the subendometrial space of the uterine treatment served as the control group (control group, N ¼
cavity, which is the niche area for growth factor and cytokine 154). Routine standard treatment was offered to all three
production (22). We assumed that the PRP injected into the groups.
subendometrium (SE-PRP) would be more effective than The primary outcome measures were ongoing pregnancy
infusing it into the endometrial surface (intrauterine; rate (OPR), defined as pregnancy continuing beyond 20 weeks
IU-PRP). To date, as per our knowledge, no studies have per transfer cycle, and live birth rate (LBR), defined as live

2 VOL. - NO. - / - 2021

 Fertil Steril Sci®

FIGURE 1

1400 pa ents
underwent ET

1104 pa ents
296 included pa ents
excluded:
with RIF fulfilling
inclusion criteria 710 with no RIF
115 -with male factor
120-with POR
10 with systemic
diseases
pa ents who pa ents who 6 with uterine
pa ents without abnormality
underwent SE- PRP underwent IU- PRP (N-
interven ons(N-154)
(N-33) 109) 15 with PGT
20 with thin
endometrium
-10 with BMI >/30
98 - pa ents with
poor quality embryos

Flow diagram of the study participants.

Noushin. Different techniques of PRP. Fertil Steril Sci 2021.

birth of an infant after 24 weeks per transfer cycle. The sec-
ondary outcome measures were clinical pregnancy rate
(CPR) per transfer cycle, defined as ultrasonographic confir-
mation of a live intrauterine pregnancy, and miscarriage
rate (MR) until 20 weeks.
Pre-PRP preparation
For SE-PRP. Patients were asked to review in the luteal phase
of the previous cycle of ET between cycle days 21 and 24 and
were administered 300 mg of granulocyte colony-stimulating
factor (G-CSF) subcutaneously once a day for 3 days to boost
the production of cytokines and growth factors and, thus,
improve the quality of the PRP sample (23). On the next
day, blood sample was collected for PRP preparation, and 5
mg of norethisterone (Primolut N) tablet was prescribed three
times a day orally for 5 days to induce a withdrawal bleed.
Couples were advised to avoid unprotected sexual intercourse
in the treatment cycle to avoid disturbing a possible rare
pregnancy.
For IU-PRP. Once the endometrium was approximately 7
mm during the index FET preparation cycle (approximately
on days 12–14 of the cycle), 300 mg of G-CSF was adminis-
tered subcutaneously once a day for 3 days. On the next
day, blood sample was collected for PRP preparation.
PRP preparation
Under aseptic precautions, 60 mL of blood was drawn with
20-G scalp vein needle and divided into 10-mL aliquots
each into four acid–citrate–dextrose solution gel vacutainer
tubes to prevent platelet activation. Samples were centrifuged
in a refrigerated centrifuge immediately after collection at
1,200 rpm for 10 min (the soft spin)—this was to separate
the RBC from plasma. The recommended temperature during
processing was 20–22  C. Plasma was then transferred into a
sterile falcon tube and again centrifuged at 3,000 rpm for 10
min (the hard spin) to obtain platelet concentrate. The super-
natant platelet-poor plasma was discarded, and platelet pellet
formed at the bottom of the tube was collected. The platelet
pellet was then made into aliquots inside two cryowells; this
was then well capped and tightened. For platelet activation,
considering the safety and avoiding additional exogenous
chemical component in the final product, we utilized
chemical-free activation protocol using liquid nitrogen where
we subjected the platelet concentrate to two to three cycles of
freezing and thawing (24, 25).
The technique of PRP preparation, which we followed,
was standardized with the Food and Drug Administration
(FDA) approved commercially available kit (Regen PRP kit).
For all patients in the PRP groups, a baseline complete blood

VOL. - NO. - / - 2021 3

ORIGINAL ARTICLE: REPRODUCTIVE DISEASES

TABLE 1

Baseline characteristics of the three groups.

SE-PRP (N [ 55) IU-PRP (N [ 109) Control (N [ 154)
Variables Mean ± SD (95% CI) Mean ± SD (95% CI) Mean ± SD (95% CI) P value
Age (years) 32.81  4.38 32.28  4.84 33.01  4.27 .425
(29.1–34.4) (29.1–33.2) (30.4–34.4)
BMI (kg/m ) 2
26.19  0.80 26.28  0.89 26.32  0.93 .721
(25.2–26.9) (25.2–26.9) (25.4–27.1)
AMH (ng/mL) 3.67  2.32 3.87  3.03 4.14  2.62 .462
(2.7–4.3) (3.2–4.4) (3.7–4.5)
Duration of infertility (years) 8.16  2.73 5.99  1.78 5.33  1.77 .001*
(7.64–8.42) (5.72–6.35) (5.12–5.69)
WBC count (cells/mm3) 26,423  1,321 26,877  1,715 – .440
(26,378–27,077) (26,552–27,203)
Baseline platelet count (mL) 259,908  33,870 266,060  47,364 – .409
(253,477–266,338) (249,265–282,855)
Platelet count in PRP (mL) 1,232,424  137,455.22 1,254,770  123,555 – .377
(1,183,684–1,281,163) (1,231,312–1,278,228)
Endometrial thickness during FET 9.31  1.17 9.64  0.66 9.78  0.85 .072
cycle (mm) (8.71–9.92) (9.51–9.77) (9.64–9.92)
No. of previous ET attempts 4.15  1.25 4.04  0.93 4.09  0.77 .866
(3.6–4.5) (3.8–4.2) (3.9–4.2)
No. of grade 1 (good) embryos 1.30  0.46 1.27  0.44 1.21  0.37 .069
transferred per ET cycle (1.1–1.4) (1.1–1.3) (1.1–1.2)
No. of grade 2 (fair) embryos 0.70  0.45 0.73  0.43 0.79  0.40 .316
transferred per ET cycle (0.58–0.83) (0.64–0.81) (0.72–0.85)
AMH ¼ antim€ ullerian hormone; BMI ¼ body mass index; CI ¼ confidence interval; ET ¼ embryo transfer; FET ¼ frozen embryo transfer; IU-PRP ¼ platelet-rich plasma administered to the endo-
metrial surface (intrauterine); PRP ¼ platelet-rich plasma; SD ¼ standard deviation; SE-PRP ¼ platelet-rich plasma administered to the subendometrium; WBC ¼ white blood cells.
Noushin. Different techniques of PRP. Fertil Steril Sci 2021.

count was performed, which was repeated after 3 days of sys-
temic G-CSF. The WBC count increased to a range of 24,000
to 30,000 cells/mm3 after G-CSF. The platelet count before
and after enrichment with PRP was checked (Table 1).
Platelet-rich plasma was administered within 10 min of
preparation.
PRP administration technique
Group: SE-PRP (N-55). Under aseptic precautions and
conscious sedation, PRP was administered into the subendo-
metrium using an oocyte recovery needle (18 G, 330 mm)
(Wallace, CooperSurgical) threaded into the ET catheter (Wal-
lace, Smiths Medical, Czech Republic) transvaginally under
USG guidance. A volume of approximately 2 mL of PRP
each was injected into the subendometrial space in the ante-
rior and posterior walls of the uterus. In 2D USG, the suben-
dometrial space is best visualized in the sagittal view. It has
two distinctive structures: basal endometrium visualized as
a continuous, uninterrupted hyperechoic line, and in practical
terms, it represents the endometrial–myometrial interface and
inner myometrium, which is seen as an uninterrupted hypo-
echoic band or ‘‘halo’’ that encircles the endometrium (26).
Group: IU-PRP (N-109). The syringe containing PRP was
connected to the ET catheter, and approximately 1 mL of
PRP was infused into the uterine cavity via ET catheter (Wal-
lace) under transabdominal USG guidance.
FET protocol
The endometrium was prepared by hormone replacement
treatment protocol. Estradiol valerate (Progynova; Bayer
Schering Pharma) was started on day 2 or 3 of the cycle at
a dose of 2 mg four times a day orally and subsequently,
and the dose was adjusted according to the response of the
endometrium to a maximum dose of 12 mg per day.
Follow-up transvaginal scan was performed to assess the
endometrial thickness from day 12 of the treatment cycle.
Once the endometrium was R8 mm, 400 mg of vaginal
micronized progesterone (Susten 400 mg capsule, Sun
Pharma) twice daily and 10 mg of oral dydrogesterone (Du-
phaston, Abbott India Ltd.) three times a day were started as
per the hospital protocol for luteal phase support, and estra-
diol valerate was continued.
Two embryos of day 3 developmental stage with at least
one morphologically good-quality or grade 1 embryo were
transferred. Grade 1 (good-quality) embryos were those hav-
ing <10% fragmentation and a cell number of >7 on day 3
with no multinucleation. Grade 2 (fair) embryos referred to
grade 1 embryos that had 10%–25% fragmentation. The em-
bryos were tagged as grade 3 when the cell number was <7 on
day 3 or there was >25% fragmentation or evidence of multi-
nucleation, as per the Istanbul consensus (27). The embryos
were transferred under USG guidance by a senior consultant
in reproductive medicine on day 4 of progesterone. Patients
were excluded from the study if the endometrial thickness
was <8 mm during the FET preparation or if there were no
grade 1 (good-quality) embryos available for ET. Luteal phase
support was continued until 10 weeks if pregnant. Transvagi-
nal ultrasonography was performed 4 weeks after the ET to
confirm viable intrauterine pregnancy.
Statistical analysis
Statistical analysis was performed using SPSS (version 20).
For normally distributed data, mean and standard deviation

4 VOL. - NO. - / - 2021

 Fertil Steril Sci®

TABLE 2

Comparison of outcome variables.

Variables SE-PRP (N [ 55) IU-PRP (N [ 109) Control (N [ 154) P value
beta hCG þve per ET cycle 36 (65.65%) 70 (64.2%) 68 (44.2%) .002*
Clinical pregnancy per ET cycle 28 (51%) 57 (52.3%) 52 (33.8%) .006*
Ongoing pregnancy/ birth per ET cycle 22 (40%) 45 (41.3%) 34 (22.1%) .004*
Miscarriage per ET cycle 14 (25.45%) 25 (22.23%) 34 (22.07%) .580
bhCG ¼ beta hCG; ET ¼ embryo transfer; IU-PRP ¼ platelet-rich plasma administered to the endometrial surface (intrauterine); SE-PRP ¼ platelet-rich plasma administered to the subendometrium.
Noushin. Different techniques of PRP. Fertil Steril Sci 2021.

were used to describe data location and dispersion. Compari-

FIGURE 2
son between the groups was evaluated using the chi-square
test and ANOVA. Multiple comparison analysis was per-
80
formed using post hoc ANOVA. Statistical significance was
set at P< .05. Logistic regression analysis was performed to 70 66.7 64.2
evaluate the effect of variables on LBR/OPR.
60
51.5 52.3
50
44.2
Percentage
41.3
39.4
40
RESULTS 33.8 27.27
Baseline characteristics other than the duration of infertility 30 SE-PRP
were similar in all three groups (Table 1). The platelet count 22.1 22.23 IU-PRP
22.07
at baseline, WBC count, PRP preparation technique, and 20 CONTROL
FET protocol were standardized and the same in all the
10
groups.
Table 2 and Figure 2 illustrate the comparison of outcome 0
variables. Multiple comparisons between groups are pre- Beta Positve Clinical Ongoing Miscarriage
sented in Table 3. While the OPR/LBR per transfer cycle per embryo pregnancy per pregnancy/
transfer embryo Live birth per
were higher in the SE-PRP and IU-PRP groups than that in transfer embryo
the control group, these were similar between the SE-PRP transfer
and IU-PRP groups. The data on beta hCG (bhCG) þve rate variables

and CPR per ET cycle showed a similar trend with a higher
rate in the SE-PRP and IU-PRP groups than that in the con-
trols. No statistical difference in the CPR was noted between
the SE-PRP and IU-PRP groups. The MR was similar in all
three groups. Table 4 shows data of logistic regression anal-
ysis evaluating the effect of variables including age, body
mass index, antim€ ullerian hormone (AMH), number of previ-
ous FET cycles, number of embryos transferred, number of
grade 1 (good) and 2 (fair) embryos transferred, and PRP treat-
ment on the OPR/LBR outcome. In the univariate analysis, age
and PRP treatment were the significant variables that influ-
enced the outcome. In the multivariate analysis, after control-
ling for age and other relevant clinical variables, both SE-PRP
and IU-PRP were still influencing the outcome favorably
(Table 4).
Time duration between IU-PRP and ET was 3-7 days
(mean -4.34) and for SE-PRP it was 22-36 days (mean-
26.18). None of the patients in the SE-PRP or IU-PRP group
developed any adverse reaction to PRP during or after the
procedure. Similarly, none in the SE-PRP or IU-PRP group
developed any infection or bleeding post procedure. SE-PRP
was administered under conscious sedation as a day-care pro-
cedure with most patients being able to ambulate indepen-
dently immediately post procedure.
Comparison of outcome variables.
Noushin. Different techniques of PRP. Fertil Steril Sci 2021.
DISCUSSION
The data in the study indicate that PRP treatment, adminis-
tered through either the subendometrial or intrauterine route,
appears to improve OPR/LBR during FET cycles in women
with a history of RIF. To the best of our knowledge, this is
the first study reporting the effectiveness of activated PRP
administered to either the subendometrium or endometrial
surface (intrauterine) in unexplained RIF. However, the data
did not show any advantage of using the subendometrial
route over the lesser invasive intrauterine administration of
PRP treatment.
Our findings of increased rates of biochemical pregnancy,
clinical pregnancy, and ongoing pregnancy or live birth in the
IU-PRP group are in accordance with the systematic review and
meta-analysis published by Maleki-Hajiagha et al. (17) in 2020,
involving seven studies showing the positive benefit of PRP,
although the studies were of heterogeneous design and popula-
tion. Quite a few researchers have studied the effectiveness of

VOL. - NO. - / - 2021 5

ORIGINAL ARTICLE: REPRODUCTIVE DISEASES

TABLE 3 TABLE 4

Multiple comparisons to evaluate the statistical difference between Logistic regression analysis to evaluate the effect of variables
each group. including platelet-rich plasma treatment on ongoing pregnancy
rate/live birth rate.
Variables Group Groups P value
Univariate analysis
bhCG þve per ET cycle SE-PRP IU-PRP .792
Control .019* Variables Odds ratio 95% CI P value
IU-PRP SE-PRP .792
Control .001* Age (years) 0.945 (0.895–0.999) <.05
Clinical pregnancy per ET SE-PRP IU-PRP .936 AMH (ng/mL) 1.064 (0.978–1.156) .15
cycle Control .049* BMI (kg/m2) 0.773 (0.589–1.015) .06
IU-PRP SE-PRP .936 No. of embryos transferred 0.810 (0.561–1.171) .26
Control .002* No. of grade 1 embryos 1.188 (0.691–2.040) .533
Ongoing pregnancy/live SE-PRP IU-PRP .846 transferred
birth per ET cycle Control .038* No. of grade 2 embryos 0.842 (0.490–1.44) .516
IU-PRP SE-PRP .846 transferred
Control .022* Previous FET attempts 0.842 (0.644–1.101) .21
SE-PRP group 2.353 (1.216–4.554) <.01
bhCG ¼ beta hCG; ET ¼ embryo transfer; IU-PRP ¼ platelet-rich plasma administered to the
endometrial surface (intrauterine); SE-PRP ¼ platelet-rich plasma administered to the
IU-PRP group 2.482 (1.448–4.254) <.01
subendometrium.
Multivariate analysis
Noushin. Different techniques of PRP. Fertil Steril Sci 2021.
Variables Odds ratio 95% CI P value

PRP to improve reproductive outcome in patients with impaired Age (years) 0.957 (0.904–1.013) .13
BMI (kg/m2) 0.798 (0. 602–1.057) .12
endometrial receptivity (28), which began with Chang and his No. of embryos transferred 0.772 (0.522–1.143) .20
colleagues in 2015, who first proposed this treatment for refrac- Previous FET attempts 0.859 (0. 655–1.128) .27
tory thin endometrium (11, 16, 29). The randomized controlled No. of grade 1 embryos 0.653 (0.313–1.361) .255
transferred
trial (RCT) by Obidniak et al. (30) also found higher CPR in the No. of grade 2 embryos 1.531 (0.735–2.650) .269
autologous IU-PRP group compared with that no treatment transferred
group (53% vs. 24.4%) in patients with RIF. However, a few SE-PRP group 2.502 (1.265–4.94) <.01
studies did not find a significant improvement with PRP treat- IU-PRP group 2.388 (1.381–4.130) <.01
AMH ¼ antim€ ullerian hormone; BMI ¼ body mass index; CI ¼ confidence interval;
ment (18). This inconsistency is mainly because there is FET ¼ frozen embryo transfer; IU-PRP ¼ platelet-rich plasma administered to the endometrial
currently no consensus regarding the ideal method of PRP surface (intrauterine); SE-PRP ¼ platelet-rich plasma administered to the subendometrium.

preparation as the majority of the studies published did not Noushin. Different techniques of PRP. Fertil Steril Sci 2021.

mention the platelet and WBC quantification, details of meth-

odology, centrifugation, and activation method. It has been
seen that the double centrifugation procedure, as performed
in our study, renders the highest output, and this may be a fac-
tor influencing the outcome (31).
In our study, there was no patient in both PRP groups
without four to five times improvement in their platelet count
from the baseline, thus potentially optimizing the benefits of
PRP (32). To prevent the premature unintentional platelet
activation, during the processing, the temperature was main-
tained at 20–22  C, and, we used a large-bore needle for blood
collection (33). The rationale behind the activation of PRP in
liquid nitrogen was that with sudden heat shock, the alpha
granules in platelets break down, releasing the growth factors
that will be more profound than PRP without platelet activa-
tion (34). As the active secretion of growth factors by platelets
begins within 10 min of activation, we administered PRP into
the cavity within 10 min of preparation (35). Our PRP sample
was leucocyte-rich as we utilized the buffy coat, which was
rich in leukocytes. There is a school of thought that leucocytes
increase inflammation, which is an essential step in the heal-
ing process especially against infection and clearing of tissue
debris (36, 37). Several studies have quoted a similar observa-
tion that leucocyte-rich PRP showed positive effects on endo-
metrial healing, receptivity, and implantation (38). Since, to
date, no well-designed studies are published on the use of
leukocyte-poor or pure PRP on endometrial receptivity, its
beneficial effect needs to be explored in future.
There is a theoretical risk that after intrauterine infusion,
a part of PRP may be flushed off, and the quantity retained in
the cavity for the actual action may be lesser than that admin-
istered. Additionally, with intrauterine infusion, the absorp-
tion rate is also questionable. Therefore, we tried a novel
technique of administering PRP into the subendometrial
space. The physiological rationale for this is for better local
absorption considering the fact that the subendometrium
and basal endometrium are the niche sites for growth factor
production, regeneration, and vascularity (17, 22). However,
we did not see a benefit with the use of subendometrial route
over the intrauterine route, and therefore, the use of more
invasive subendometrial PRP can be restricted. Due to the
invasive nature and the risk of damaging the growing endo-
metrium, SE-PRP cannot be administered in the index cycle of
FET preparation. This variable timing of PRP administration,
with IU-PRP administered close to ET and SE-PRP adminis-
tered much more remotely to the ET date, would make it un-
fair to unconditionally compare between the two, and this
could have potentially influenced the outcome. To date, there
are no comparative studies evaluating different techniques of
PRP administration. Therefore, we have no studies to compare
our finding of subendometrial PRP being less effective than
routine intrauterine PRP. As an additional intervention,
G-CSF was administered for the study group during PRP
preparation, while the control group did not receive it; the

6 VOL. - NO. - / - 2021


intention for offering G-CSF was to boost the cytokine and
growth factor production and, thus, improve the quality of
PRP sample rather than specifically for RIF. Majority of the
published literature utilized intrauterine G-CSF rather than
the subcutaneous method for better local uterine action in
women with thin endometrium or RIF. Although to date there
is no robust conclusion on the role of G-CSF in RIF (39), we
acknowledge this as the limitation of the study.
The MR in all three groups were found to be similar. While
PRP may have benefited in supporting implantation, it has not
shown a similar effect in reducing the miscarriage. However,
considering the most likely reason for miscarriage being chro-
mosomal abnormalities, it is a biological plausibility that PRP is
of no benefit in improving miscarriage. We have not seen any
adverse events or side effects with the use of PRP through both
routes, although women in the SE-PRP group needed to have
the day-care procedure under conscious sedation. Our unit
runs a specialist referral clinic for RIF, and this accounts for
higher incidence of RIF in our patient population. The strength
of our study is that we utilized a novel route of administration
(subendometrial), albeit its invasive nature. Further, the sample
size in the IU-PRP group and controls were larger than those in
the reported studies. Our PRP preparation technique and meth-
odology were standardized with FDA-approved kits, which
were logistically affordable compared with commercially avail-
able kits, and the activation of PRP was free of chemical
composition. While we utilized a prospective design, our study
is limited by the fact that it is not an RCT. Therefore, a large,
prospective, double-blinded, placebo-controlled RCT is war-
ranted to generate further robust data before recommending
whether these interventions can be used in routine practice to
benefit those with RIF.
In conclusion, PRP treatment seems to improve OPR/LBR
during FET cycles in women with a history of RIF. However,
the data did not show any advantage of using the subendometrial
route over lesser invasive intrauterine administration of PRP
treatment. While the data from the study indicates that PRP treat-
ment has the potential to increase the take-home baby rate in
women with RIF, further large RCT is warranted to generate
high-quality evidence on its regular use in cases of RIF.
Acknowledgment: The authors thank Mr. Tittu Raju for the
statistical analysis.
REFERENCES
1. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treat-
ment of repeated implantation failure following IVF-ET. Hum Reprod 2006;
21:3036–43.
2. Coughlan C, Ledger W, Wang Q, Liu F, Demirol A, Gurgan T, et al. Recurrent
implantation failure: definition and management. Reprod Biomed Online
2014;28:14–38.
3. Das M, Holzer HE. Recurrent implantation failure: gamete and embryo fac-
tors. Fertil Steril 2012;97:1021–7.
4. Mahajan N. Endometrial receptivity array: clinical application. J Hum Reprod
Sci 2015;8:121–9.
5. Barash A, Dekel N, Fieldust S, Segal I, Schechtman E, Granot I. Local injury to
the endometrium doubles the incidence of successful pregnancies in pa-
tients undergoing in vitro fertilization. Fertil Steril 2003;79:1317–22.
6. Madkour W, Noah B, Zaheer H, Al-Bahr A, Abdelhamid A, Shaeer M, et al.
Does sequential embryo transfer improve pregnancy rate in patients with
VOL. - NO. - / - 2021
Fertil Steril Sci®
repeated implantation failure? A randomized control study. Middle East Fer-
til Soc J 2015;2.
7. Nobijari FF, Arefi SS, Moini A, Taheripanah R, Fazeli E, Kharazi H, et al. Endo-
metrium immunomodulation by intrauterine insemination administration of
treated peripheral blood mononuclear cell prior frozen/thawed embryos in
patients with repeated implantation failure. Zygote 2019;27:214–8.
8. Nyborg K, Kolte A, Larsen E, Christiansen O. Immunomodulatory treatment
with intravenous immunoglobulin and prednisone in patients with recurrent
miscarriage and implantation failure after in vitro fertilization/intracytoplas-
mic sperm injection. Fertil Steril 2014;102:1650–5.
9. Katzorke N, Vilella F, Ruiz M, Kru n C. Diagnosis of endometrial-
€ssel J, Simo
factor infertility: current approaches and new avenues for research. Geburt-
shilfe Frauenheilkd 2016;76:699–703.
10. Lee JW, Kwon OH, Kim TK, Cho YK, Choi KY, Chung HY, et al. Platelet-rich
plasma: Quantitative assessment of growth factor levels and comparative
analysis of activated and inactivated groups. Arch Plast Surg 2013;40:530–5.
11. Chang Y, Li J, Chen Y, Wei L, Yang X, Shi Y, et al. Autologous platelet-rich
plasma promotes endometrial growth and improves pregnancy outcome
during in vitro fertilization. Int J Clin Exp Med 2015;8:1286–90.
12. Aghajanova L, Houshdaran S, Balayan S, Irwin J, Huddleston H, Giudice L.
Platelets for endometrial regeneration: a novel approach. Fertil Steril
2016;106:e82.
13. Dhillon RS, Schwarz EM, Maloney MD. Platelet-rich plasma therapy: future
or trend? Arthritis Res Ther 2012;14:219.
14. Nazari L, Salehpour S, Hoseini S, Zadehmodarres S, Ajori L. Effects of autol-
ogous platelet-rich plasma on implantation and pregnancy in repeated im-
plantation failure: a pilot study. Int J Reprod Biomed 2016;14:625–8.
15. Farimani M, Poorolajal J, Rabiee S, Bahmanzadeh M. Successful pregnancy
and live birth after intrauterine administration of autologous platelet-rich
plasma in a woman with recurrent implantation failure: a case report. Int J
Reprod Biomed 2017;15:803–6.
16. Tandulwadkar SR, Naralkar MV, Surana AD, Selvakarthick M, Kharat AH.
Autologous intrauterine platelet-rich plasma instillation for suboptimal
endometrium in frozen embryo transfer cycles: a pilot study. J Hum Reprod
Sci 2017;10:208–12.
17. Maleki-Hajiagha A, Razavi M, Rouholamin S, Rezaeinejad M,
Maroufizadeh S, Sepidarkish M. Intrauterine infusion of autologous
platelet-rich plasma in women undergoing assisted reproduction: a system-
atic review and meta-analysis. J Reprod Immunol 2020;137:103078.
18. Madhavan A, Naidu P, Rani KK, Kaur J, Mahajan N. Intrauterine autologous
platelet-rich plasma therapy to improve implantation rates in patients under-
going frozen embryo transfer: a pilot study. Onco Fertil J 2018;1:81–5.
19. Garcia-Velasco J, Acevedo B, Alvarez C, Alvarez M, Bellver J, Fontes J, et al.
Response: in reference to ‘Strategies to manage refractory endometrium:
state of the art 2016’. Reprod Biomed Online 2016;33:605.
20. Amable PR, Carias RB, Teixeira MV, da Cruz Pacheco I, Correa do Amaral RJ,
Granjeiro JM, et al. Platelet-rich plasma preparation for regenerative medi-
cine: optimization and quantification of cytokines and growth factors.
Stem Cell Res Ther 2013;4:67.
21. Zwiep T, Humphrey R, Fortin D, Inculet RI, Malthaner RA. Autologous
platelet rich plasma and concentrated platelet poor plasma are safe in pa-
tients requiring lobectomies but do not reduce the duration of air leak: a ran-
domized controlled trial. Ann Surg Int 2016;2:2–11.
22. Singh N, Mohanty S, Seth T, Shankar M, Bhaskaran S, Dharmendra S. Autol-
ogous stem cell transplantation in refractory Asherman's syndrome: a novel
cell based therapy. J Hum Reprod Sci 2014;7:93–8.
23. Bendall LJ, Bradstock KF. G-CSF: From granulopoietic stimulant to bone
marrow stem cell mobilizing agent. Cytokine Growth Factor Rev 2014;25:
355–67.
24. Bielecki T, Dohan Ehrenfest DM. Platelet-rich plasma (PRP) and platelet-rich fibrin
(PRF): surgical adjuvants, preparations for in situ regenerative medicine and tools
for tissue engineering. Curr Pharm Biotechnol 2012;13:1121–30.
25. Wen J, Li HT, Li SH, Li X, Duan JM. Investigation of modified platelet-rich
plasma (mPRP) in promoting the proliferation and differentiation of dental
pulp stem cells from deciduous teeth. Braz J Med Biol Res 2016;49:e5373.
26. Naftalin J, Jurkovic D. The endometrial-myometrial junction: A fresh look at a
busy crossing. Ultrasound Obstet Gynecol 2009;34:1–11.
7
ORIGINAL ARTICLE: REPRODUCTIVE DISEASES
27. Alpha Scientists in Reproductive Medicine, ESHRE Special Interest Group
Embryology. Istanbul consensus workshop on embryo assessment: proceed-
ings of an expert meeting. Reprod Biomed Online 2011;22:632–46.
28. Kim H, Shin JE, Koo HS, Kwon H, Choi DH, Kim JH. Effect of autologous
platelet-rich plasma treatment on refractory thin endometrium during the
frozen embryo transfer cycle: a pilot study. Front Endocrinol 2019;10:61.
29. Zadehmodarres S, Salehpour S, Saharkhiz N, Nazari L. Treatment of thin
endometrium with autologous platelet-rich plasma: a pilot study. JBRA
Assist Reprod 2017;21:54–6.
30. Obidniak D, Gzgzyan A, Feoktistov A, Niauri D. Randomized controlled trial
evaluating efficacy of autologous platelet-rich plasma therapy for patients
with recurrent implantation failure. Fertil Steril 2017;108:e370.
31. Tamimi FM, Montalvo S, Tresguerres I, Blanco Jerez L. A comparative study
of 2 methods for obtaining platelet-rich plasma. J Oral Maxillofac Surg 2007;
65:1084–93.
32. Tetlow RL, Richmond I, Manton DJ, Greenman J, Turnbull LW, Killick SR. His-
tological analysis of the uterine junctional zone as seen by transvaginal ultra-
sound. Ultrasound Obstet Gynecol 1999;14:188–93.
33. Macey M, Azam U, McCarthy D, Webb L, Chapman ES, Okrongly D, et al.
Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine,
8 VOL. - NO. - / - 2021
and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120
hematology system. Clin Chem 2002;48:891–9.
34. Park HB, Yang JH, Chung KH. Characterization of the cytokine profile of
platelet rich plasma (PRP) and PRP-induced cell proliferation and migration:
upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells. Korean J
Hematol 2011;46:265e73.
35. Marx RE. Platelet-rich plasma (PRP): What is PRP and what is not PRP?
Implant Dent 2001;10:225–8.
36. Bielecki TM, Gazdzik TS, Arendt J, Szczepanski T, Krol W, Wielkoszynski T. Anti-
bacterial effect of autologous platelet gel enriched with growth factors and
other active substances: an in vitro study. J Bone Joint Surg Br 2007;89:417–20.
37. Martin P, D’Souza D, Martin J, Grose R, Cooper L, Maki R, et al. Wound heal-
ing in the PU.1 null mouse–tissue repair is not dependent on inflammatory
cells. Curr Biol 2003;13:1122–8.
38. Cousins FL, Kirkwood PM, Saunders PT, Gibson DA. Evidence for a dynamic
role for mononuclear phagocytes during endometrial repair and remodel-
ling. Sci Rep 2016;6:36748.
39. Kamath MS, Kirubakaran R, Sunkara SK. Granulocyte-colony stimulating
factor administration for subfertile women undergoing assisted reproduc-
tion. Cochrane Database Syst Rev 2020;1:CD013226.
 Fertil Steril Sci®

1 A comparative evaluation of subendometrial and

intrauterine platelet-rich plasma treatment for
women with recurrent implantation failure
M. A. Noushin, M. Ashraf, C. Thunga,
S. Singh, S. Singh, R. Basheer, R. Ashraf, and
K. Jayaprakasan
Thrissur, Kerala, India; and Derby, United
Kingdom

VOL. - NO. - / - 2021

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