Intraovarian insertion of autologous platelet growth factors as cell-free concentrate: Fertility recovery and first unassisted conception with term delivery at age over 40

Abstract

Int J Reprod Biomed [2020] :: Background: Use of autologous platelet-rich plasma (PRP) as an ovarian treatment has not been standardized and remains controversial. Case Presentation: A 41½ year old with diminished ovarian reserve (serum AMH=0.163mg/mL) and history of 10 unsuccessful IVF cycles presented for reproductive endocrinology consult. She and her partner declined donor oocyte IVF. They both were in good general health and laboratory tests were unremarkable, except for mild thrombocytosis (platelets=386K; normal range 150K-379K) discovered in the female. Here the patient underwent intraovarian injection of fresh platelet-derived growth factor concentrate administered as an enriched cell-free substrate. Serum AMH increased by 115% within 6wks of treatment. Results: Spontaneous ovulation occurred the month after injection and serum hCG was subsequently noted at 804mIU/mL. Following an uneventful obstetrical course, a male infant was delivered at term without complication. Conclusion: This is the first description of intraovarian injection of enriched platelet-derived growth factors followed by unassisted pregnancy and livebirth. As a refinement of conventional ovarian PRP therapy, this procedure may be particularly valuable for refractory cases where prognosis for pregnancy appears especially bleak. A putative role for thrombocytosis is also viewed in parallel with mechanisms of action advanced earlier. As experience with ovarian application of autologous platelet growth factors continues, additional research will evaluate laboratory protocol/sample preparation, injection technique, and patient selection.

Full text

International Journal of Reproductive BioMedicine
Volume 18, Issue no. 12, https://doi.org/10.18502/ijrm.v18i12.8030
Production and Hosting by Knowledge E
Case Report
Intraovarian insertion of autologous platelet
growth factors as cell-free concentrate:
Fertility recovery and rst unassisted
conception with term delivery at age over
40
E. Scott Sills1, 2, 3 M.D., Ph.D., Natalie S. Rickers1, 3 LVN, Samuel H. Wood2, 3
M.D. Ph.D.
1Reproductive Biology Group IVF, FertiGen CAG; San Clemente, California USA.
2Department of Obstetrics and Gynecology, Palomar Medical Center; Escondido, California USA.
3Gen 5 Fertility Center; San Diego, California USA.
Abstract
Background: The use of autologous platelet-rich plasma as an ovarian treatment has
not been standardized and remains controversial.
Case Presentation: A 41½-year old woman with diminished ovarian reserve (serum anti-
Müllerian hormone = 0.163 mg/mL) and a history of 10 unsuccessful in vitro fertilization
cycles presented for reproductive endocrinology consult. She and her partner declined
donor oocyte in vitro fertilization. They were both in good general health and laboratory
tests were unremarkable, except for mild thrombocytosis (platelets = 386K; normal
range 150-379K) discovered in the female. The patient underwent intraovarian injection
of fresh platelet-derived growth factor concentrate administered as an enriched cell-
free substrate. Serum anti- Müllerian hormone increased by 115% within 6 wks of
treatment. Spontaneous ovulation occurred the month after injection and subsequently
the serum human chorionic gonadotropin was noted at 804 mIU/mL. Following an
uneventful obstetrical course, a male infant was delivered at term without complication.
Conclusion: This is the rst description of intraovarian injection of enriched platelet-
derived growth factors followed by unassisted pregnancy and live birth. As a renement
of conventional ovarian platelet-rich plasma therapy, this procedure may be particularly
valuable for refractory cases where prognosis for pregnancy appears especially bleak.
A putative role for thrombocytosis is also viewed in parallel with mechanisms of action
as advanced earlier. With continued experience in ovarian application of autologous
platelet growth factors, additional research will evaluate laboratory protocol/sample
preparation, injection technique, and patient selection.
Key words: Ovarian rejuvenation, Platelet-rich plasma, Cytokines, Infertility, IVF.
How to cite this article:Sills ES, Rickers NS, Wood SH. “Intraovarian insertion of autologous platelet growth factors as cell-free concentrate: Fertility recovery
and rst unassisted conception with term delivery at age over 40,” Int J Reprod BioMed 2020; 18: 1081–1086. https://doi.org/10.18502/ijrm.v18i12.8030 Page 1081
Corresponding Author:
E. Scott Sills, P.O. Box 73910
San Clemente, California
92673 USA.
Tel: (+1) 949 -899-5686
Email: ess@prp.md
Received 18 July 2020
Revised 9 September 2020
Accepted 8 November 2020
Production and Hosting by
Knowledge E
Sills et al. This article is
distributed under the terms
of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that
the original author and source
are credited.
Editor-in-Chief:
Aatoonian Abbas M.D.

International Journal of Reproductive BioMedicine Sills et al.
1. Introduction
It is well-known that as women age, both the
quality and the quantity of eggs decline; the low
ovarian reserve observed among older infertile
patients occurs as an expected physiological
consequence of normal ovarian senescence.
In such cases, even the use of high-dose
gonadotropin protocols is generally futile, leaving
oocyte donation/IVF as the only clinically effective
treatment (1-3).
As an investigational alternative to egg donation,
the surgical placement of autologous platelet-
rich plasma (PRP) into ovarian tissue rst began
to attract attention in 2016 (4). This pioneering
technique of ovarian “rejuvenation” was followed
by two publications describing similar use of PRP
for poor-prognosis patients as a precursor to IVF.
Specically, four patients with undetectable or
very low ovarian reserve (mean age 42 years)
who had planned for donor egg treatment instead
underwent the PRP treatment; all four patients
developed blastocysts from their oocytes (5), one
of them has since undergone thaw, transfer, and
had a healthy term delivery. Experts in Greece
also described three poor-responder IVF patients
(mean age 38 years) with similar “revolutionary”
responses (6). At least one patient who produced
only embryos with genetic errors was able to
achieve “ploidy rescue” following intraovarian
injection of platelet-derived growth factors (PDGFs)
before IVF, culminating in successful term live birth
(7). Ovarian PRP has been formally evaluated in
a descriptive pilot study including >150 patients,
where no signicant change was observed in the
serum AMH of most patients (8). However, the
measured response rate after ovarian PRP (28%)
approximates the overall IVF pregnancy rate in the
USA. While ovarian treatment with growth factors
has generally been framed as a precursor to IVF (5),
almost no data exist on the reproductive outcome
in the absence of IVF. Here, intraovarian injection of
enriched, cell-free platelet growth factors followed
by healthy term delivery - with no gonadotropins or
IVF - is presented.
2. Case Presentation
A 41½-year old woman presented with her
husband (aged 41) for reproductive endocrinology
consultation. They were both in good general
health and took no regular medication. Her
past surgical history was signicant for an
uncomplicated laparoscopic myomectomy in
2018. The assessment of endometrial cavity
contour after the procedure was normal. At age
38, the patient was provisionally diagnosed with
primary ovarian insufciency (POI) based on
repeatedly elevated levels of follicle-stimulating
hormone (FSH) and “undetectable” anti-Müllerian
hormone (AMH). The remainder of the work-
up in this case was essentially unremarkable,
including a normal endocrine prole and negative
pregnancy test, although a mild thrombocytosis
(platelets = 386K; normal range 150-379K)
was discovered in the female. Moreover, two
pregnancies were established without medical
assistance >5 yrs ago, but both were electively
terminated without complication. The couple had
initiated at least 10 IVF cycles elsewhere before
consultation, however, none were successful; chart
review attributed these failures to poor follicular
response, “empty follicles,” fertilization failure,
or culture arrest. Additional IVF attempts were
discouraged, and for personal reasons the couple
declined donor egg IVF.
Here, the patient was counseled and a written
informed consent was obtained for the injection
of enriched autologous PDGFs into both ovaries.
This treatment was offered as an extension of
a previous IRB-approved prospective clinical
trial (8). PDGFs were isolated rst via obtaining
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International Journal of Reproductive BioMedicine Fertility recovery with platelet growth factors
autologous PRP activated by calcium gluconate
as previously described (8), followed by additional
enrichment by centrifugation at 250 g ×15 min
with phosphate-buffered saline (Thermo Fisher
Scientic, Carlsbad Calif USA) irrigation of the
platelet pellet ×3. Thereafter, the resuspended
platelet pellet was next processed through
a centrifugation sequence (300 ×g for 10
min, 2,000 ×g for 10 min) to subtract debris,
based on prior protocols (9-11). The resulting
cell-free supernatant was maintained fresh at
room temperature for ovarian insertion. After
processing, a volume of approximately 1.5 mL
was injected into ovarian stroma (subcapsular)
under direct transvaginal ultrasound guidance
with instrumentation as for conventional PRP
dosing (5). The procedure was well tolerated
with no complications, completed in <10 min,
and required no anesthesia or sedation. Using a
uniform assay (12), the serum AMH was checked
again the following month, and the level had
increased from 0.163 ng/mL pretreatment to
0.352 ng/mL six weeks later (Figure 1). Ovulation
occurred without medical assistance and serum
human chorionic gonadotropin was subsequently
noted at 804 mIU/mL. Her prenatal course was
uneventful and she delivered a healthy 3,740
gr male infant without complication by elective
cesarean at 39 weeks gestation. Both the mother
and the baby continue to do well.
Figure 1. Serum AMH levels measured before versus after bilateral intraovarian injection of platelet-derived growth factors.
Approximately six weeks after the treatment (blue arrow), an unassisted pregnancy was conrmed (green arrow). Baseline
thrombocytosis is depicted at left (red arrow) to show nominally elevated pretreatment platelet concentration relative to the
expected reference range (vertical bar). AMH: Anti-mullerian hormone; INJ: Intraovarian injection of enriched platelet growth
factors; hCG: Human chorionic gonadotropin; PLT: Platelet concentration; Dashed/solid line: Estimated/veried data.
2.1. Ethical consideration
Written informed consent was obtained from the
patient, who read and approved the manuscript
before publication.
3. Discussion
For both patients and providers, the problem of
recurrent IVF failure is difcult and usually leads
to discussion about donor oocytes. Although
https://doi.org/10.18502/ijrm.v18i12.8030 Page 1083

International Journal of Reproductive BioMedicine Sills et al.
this approach is an established component of
advanced fertility treatment since rst introduced in
the 1980s (13, 14), it is still sometimes unacceptable
to some patients. For this reason, any advances
to open safe and effective opportunities for
patients would be welcomed. While successful
pregnancy has been reported even when POI
has been validated (15), the numerous IVF failures
experienced by our patient focused renewed
attention on intraovarian injection of autologous
platelet-derived cytokines. Although no consensus
exists regarding any preferred PRP sample
preparation or injection technique, there is even
less agreement regarding an optimal methodology
for the newer approach for incubation and/or
isolation of platelet growth factors.
As summarized in Figure 2, selected
platelet releasate components are shown with
depleted platelets (DEP). These include TGF-β,
a transcription activator modulating genes for
differentiation, chemotaxis, and proliferation and
activation of immune system cells; vascular
endothelial growth factor, a signal protein
stimulating blood vessel formation; insulin-like
growth factors (1 and 2), proteins required for
cell stimulation; PDGF, critical to blood vessel
growth from adjacent capillaries, mitogenesis,
and proliferation of mesenchymal cells including
broblasts, osteoblasts, tenocytes, vascular
SMCs, and mesenchymal stem cells; Interleukin-
1β (IL-1β), an inammatory marker involved in
cell growth, differentiation, and programmed
death; Interleukin-8 which initiates angiogenesis,
perfusion, and movement to injury/infection sites;
epidermal growth factor, a key messenger in
cell proliferation, differentiation, and survival;
as well as basic broblast growth factor, a
mediator with mitogenic and cell survival
activities including embryonic development,
cell growth, morphogenesis, and tissue
repair.
Placing such growth factors within ovarian tissue
may potentiate higher AMH output and improve IVF
response in several ways (16). One possibility is that
any follicle emerging after intraovarian injection
of these growth factors was merely latent, not
completely absent. Perhaps more controversially,
PDGFs could engage with uncommitted ovarian
stem cells (17) and work along multiple signaling
pathways to evoke differentiation to de novo
oocytes. Indeed, PRP has been shown to induce
proliferation of some cell populations to improve
stemness and to enhance in vitro expression of
receptivity markers (18, 19). Since these factors
also have angiogenic properties, it is plausible that
improving capillary ow and thus tissue oxygen
delivery might induce benecial ovarian effects
after injection (20).
While it is tempting to ascribe any effects
observed here to platelet cytokines, it is not
possible to separate this component from the
injection process itself where ovarian micro-
puncture alone might be therapeutic (21, 22).
However, we believe the link between the
treatment and subsequent pregnancy here is
supported by the brief interval between treatment
and pregnancy, the absence of any other therapy,
the serum AMH pattern after injection, and
thrombocytosis. This latter issue draws notice to
the role of platelet dynamics when ovarian PRP and
related treatments are critically assessed; ambient
platelet count has been identied as a modulator
of AMH response independent of patient age,
infertility duration, or pre-injection AMH level (5,
8).
We agree that limitations exist whenever
case data are considered. For example,
the marked uptick in serum AMH and the
favorable reproductive outcome attained
here after treatment are associative and not
necessarily causative. Additional study should
help clarify signaling pathways involved in
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International Journal of Reproductive BioMedicine Fertility recovery with platelet growth factors
follicular development, thereby providing potential
techniques to make pregnancy possible even
for older patients with low or absent ovarian
reserve.
Figure 2. Outline comparing standard platelet-rich plasma (PRP) versus enriched platelet factors (EPF) ovarian treatment.
Conict of Interest
ESS and SHW have received a provisional U.S.
patent for the process & treatment of ovarian
disorders using platelet cytokine derivatives. NSR
has no conicts to disclose.
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