Keywords
ovary - premature ovarian insufficiency - cyclophosphamide - in vitro fertilization
- platelet-rich plasma
Palavras-chave
ovário - insuficiência ovariana prematura - ciclofosfamida - fertilização in vitro
- plasma rico em plaquetas
Introduction
While ovarian reserve is defined as the number of follicles/oocytes present in the
ovaries, premature ovarian insufficiency (POI) is defined as a decrease in ovarian
functions and oocyte reserve before the age of 40.[1] The prevalence of POI is estimated to be around 1 to 3% among women when the general
population is evaluated.[2] Most POIs are classified as idiopathic.[3] However, the pathophysiology of POI is thought to be related to genetic factors,
radiotherapy, and chemotherapy factors, autoimmune disorders, and infections.[3] Premature ovarian insufficiency results in premature loss of ovarian function, major
health problems, and infertility, especially as a result of the decreased number of
oocytes in the ovaries due to accelerated atresia. In case of sufficient residual
ovarian reserve, in vitro fertilization (IVF) with autologous oocytes obtained by
ovarian stimulation is an effective treatment for women with POI.[4]
Platelet-rich plasma (PRP) is an autologous product rich in many growth factors, such
as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, and
vascular endothelial growth factor (VEGF).[5]
[6] Growth factors in PRP stimulate chemotaxis, proliferation, and differentiation of
stem cells and angiogenesis in a way that accelerates tissue repair.[7]
[8] Platelet-rich plasma, which is an inexpensive product compared with many other agents,
has many advantages, such as being easy to obtain and having an antimicrobial effect
as well as being an autologous product.[9]
Alkylating chemotherapy agents such as cyclophosphamide (CYC), which are highly gonad-toxic,
cause a decrease in ovarian function and have detrimental effects on the female reproductive
organs.[10] These effects of CYC are primarily due to the inhibition of DNA synthesis and function
and induction of DNA damage. Cyclophosphamide has been shown to reduce primitive follicles,
oocytes, and granulosa cells on eggs by inducing apoptosis, inhibiting angiogenesis,
thus causing ovarian atrophy.[11]
Pathological changes in CYC-generated POI patterns are similar to clinical observations
in POI patients, and these pathological changes in the POI model can be reversed with
drugs.[12]
Growth factors such as VEGF, EGF, PDGF, and TGF-have been shown to have protective
effects on ovarian damage.[6]
[13]
[14]
[15] Platelet-rich plasma has been found to have a predominant positive effect on ovarian
cortex volume, antral follicle number and antral follicle diameter on ovarian damage
caused by CYC.[12]
[16]
There are various medical treatments, such as immunomodulating therapies, apoptotic
inhibitors, antioxidant therapies, IVF, and embryo transfer using donor oocytes to
restore impaired ovarian function and/or restore fertility in women with POI.[17] Women with POI require significantly higher doses of exogenous gonadotropin to initiate
folliculogenesis compared with patient groups with normal ovarian reserve.[4] They commonly have a poor response to stimulation, with only four or fewer follicles
available for oocyte retrieval.[4] It seems that not every approach applied to remedy this situation can be created
as effective or guaranteed for successful management.[4] The protective and curative effect of PRP at the level of folliculogenesis in CYC-induced
ovarian damage has been shown in previous studies.[12]
[16] A recent study has shown that intraovarian injection of autologous PRP has improved
IVF results in women with primary ovarian insufficiency.[18] The aim of this study is to investigate the protective effect of PRP on in vitro
fertilization in female rats with CYC-induced ovarian damage.
Methods
The study was conducted in Sakarya University's SÜDETAM laboratory under the authority
of Sakarya University's experimental animal ethics committee on 04/11/2020 under decision
No.62. Applications for all research animals were performed according to the “The
European Commission Directive 86/609/ECC guideline” protocol. Twenty-eight adult female
Sprague-Dawley rats (weight 200–250 g; age 65–75 days) were provided by the Sakarya
University Animal Reproduction Center and housed in groups with ad libitum food and
water in the Animal Laboratory of Sakarya University. The holding room was maintained
at room temperature of 22 ± 2°C with humid conditions (45–55%) and a 12-hour light/day
cycle.
The rats were randomly divided into four different experimental (Exp.) groups:
Group I (control group) received sodium chloride 0.9% (1 mL/kg, single dose) intraperitoneal
(IP) injection on the 1st, 8th, and 15th days.
Group II (CYC group) received cyclophosphamide (CYC) (75 mg/kg, single dose) IP injection
on the 1st, 8th, and 15th, days.
Group III (CYC + PRP group) received CYC (75 mg/kg, single dose) and PRP (200 µl,
single dose) IP injection on the 1st, 8th, and 15th days.
Group IV (PRP only group) received PRP (200 µl, single dose) IP injection on the 1st, 8th, and 15th days.
The stage of the estrous cycle of the rats was determined by performing daily vaginal
smears after acclimation. Rats determined to have at least 3 consecutive 4-day estrous
cycles were prepared for in vitro fertilization (IVF). All the rats were subjected
to the IVF protocol to create hyperstimulation. On the day the stimulation was completed,
female rats were sacrificed, and their oocytes were collected.
Human tubal fluid (HTF) medium (Cat. No. 90166, Irvine Scientific, Santa Ana, CA,
USA) was used for sperm preincubation, fertilization, and embryo transfer. For sperm
preincubation, a 200 mL droplet was used. For oocyte collection and IVF, a 100 mL
volume droplet was used. Embryos were washed by passing through four such droplets.
Each droplet was placed on a 35-mm culture dish (Nunc, Cat. No.63754, Denmark), covered
with liquid paraffin oil (Cat. No. 9305, Irvine Scientific), and kept at 37°C under
5% CO2 in humidified air overnight.
The ovaries were stimulated through the IP route for both ovaries in the female rats.
For the first injection, we used an IP injection of 150 to 300 internal units (IUs)/kg
of pregnant mare serum gonadotropin (PMSG) (Chronogest/PMSG, Intervet, Istanbul, Turkey),
followed ∼ 48 hour later by 150 to 300 IUs/kg of human chorionic gonadotropin (hCG;
Gonatropin, Chorulon Intervet, Istanbul, Turkey). At 17 to 19 hours after hCG administration,
15 IUs of PMSG were administered.[19] All the rats were weighed and anesthetized by an intramuscular administration of
50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi Warner-Lambert Ilac Sanayi,
Levent, Istanbul, Turkey) and 7 mg/kg xylazine hydrochloric acid (Rompun, Bayer Sisli,
Istanbul, Turkey). After immobilizing the rats on a standard surgery board, blood
samples were collected to measure the level of serum anti-Mullerian hormone (AMH).
The aseptic technique was used to make a ventral midline incision to expose the reproductive
organs, and the oviducts were removed. In this manner, the oocytes were collected
from removed ovaries. To incubate the oocytes, HTF medium with the addition of 4 mg/ml
of human serum albumin (HSA) was cultured for 1 day before being placed in an incubator
at 37°C and 5% CO2. Culture drops were prepared as group cultures on the culture dish under mineral
oil. Fertilization, 2 washes, and culture drops were prepared in 500 µl, 150 µl, and
150 µl amounts, respectively. The oocytes and capacious sperm (approximate concentration
1 × 106 ml-1) were transferred to the fertilization drops. Then, fertilization was
checked, and the fertilized oocytes were washed and transferred to culture drops,
and the resulting embryos were monitored up to the blastocyst stage.[20]
Before the oocyte collection, a mixture of 75 mg/kg of ketamine (Ketasol, Richter
Pharma, Austria) and 10 mg/kg of xylazine (Basilazin, Bavet, Turkey) was applied intraperitoneally
to a male rat, and then the rat was euthanized. Following the euthanasia procedure,
the male reproductive system was surgically opened from the abdomen, and the left
and right epididymis were separated from the testicles and transferred to HTF medium
containing 1 ml of HTF (Cat. No. 90168, Irvine Scientific, USA) and 4 mg/ml of bovine
serum albumin (BSA). The epididymis was carefully peeled off using forceps, and the
sperm were transferred into petri dishes and incubated at 37°C for 30 minutes before
in vitro fertilization.[21]
Approximately 6.5 hours after insemination, the oocytes were washed 3 times with HTF
medium and cultured as above. At 7 to 8 hours after insemination, the oocytes were
checked for sperm penetration or pronuclear formation under an inverted microscope
to identify any polyspermic fertilization or parthenogenetic embryos (∼ 6.5% of the
total). After culturing for a further 20 hours, the numbers of 2-cell stage embryos
were counted; these were defined as fertilized embryos.
Eight mature male Sprague-Dawley rats were used to prepare PRP. Blood samples were
taken from these rats by heart puncture from the right ventricle under anesthesia
and taken into test tubes containing 3.2% sodium citrate (Merck, Darmstadt, Germany)
at the rate of 9/1 blood/citrate. After the blood samples were centrifuged at 400 × for
10 minutes, the upper part of the plasma containing the platelets and buffy coat was
transferred to another tube and centrifuged again at 800 × g for 10 minutes. This
tube contained platelet deposits and some red blood cells (an erythrocyte-platelet
cluster). By removing the upper ⅔ of the supernatant containing platelet-poor plasma,
the remaining layer (lower ⅓) was accepted as PRP. The final fraction, containing
2.4 × 106 platelets/ml, was ∼ 3.9 times larger than the blood platelet count (570,000
platelets/μl). We used fresh PRP per administration.
Anti-Mullerian hormone was quantitatively estimated in rat serum samples using enzyme-linked
immunosorbent assay (ELISA) kits (MyBioSource, Rat AMH ELISA Kit Catalog No: MBS2509909,
San Diego, California, USA).
Statistical analyses were performed using the IBM SPSS Statistics for Windows, Version
24.0 software (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk test was used to evaluate
the normal distribution of the data. For the comparison of more than two variables,
one-way analysis of variance (ANOVA) was used for normally distributed data and the
Kruskal-Wallis test was used for data that did not show normal distribution. To determine
which group was different from the others, the Tukey honestly significant difference
(HSD) test was used for variables with homogeneous variances, and the Tamhane T2 test
for non-homogeneous variables. The results are given as mean ± standard error (SE).
The statistical evaluation was considered significant when p < 0.05 for each test.
Results
The oocytes were classified as germinal vesicle (GV), metaphase I (M1), and metaphase
II (M2). To compare the meiotic progression during oocyte maturation in different
systems, the average time that each stage of nuclear progression takes was calculated.
This method was previously described by Sirard et al.[22] As a result of the statistical evaluation made in the light of this situation, it
was seen that only cyclophosphamide (CYC) application decreased the average number
of M1 and M2, increased the number of GVs, and PRP application prevented this effect
of CYC ([Fig. 1]). In the comparisons in terms of M1 and M2 numbers, it was observed that the CYC
group presented a significantly lower number than the control, CYC/PRP, and PRP groups
(for M1, respectively: p = 0.000, p = 0.029, p = 0.025; for M2, respectively: p = 0.009, p = 0.004, p = 0.000). In the evaluation made in terms of the GVs number, it was observed that
the GVs number increased in the CYC group, and the PRP application decreased the GVs
number. In the comparisons between groups, the GV value in the CYC group was significantly
higher compared with the control, CYC + PRP, and PRP groups (p = 0.001, p = 0.003, p = 0.003, respectively). When the CYC + PRP group was compared with the control and
PRP groups, there was no significant difference in terms of MI, MII, GV, and oocyte
count (p > 0.05). The average number of oocytes, fertilized oocytes and two-celled good quality
embryos belonging to the groups are presented in [Fig. 2]. The mean oocyte count was statistically significantly lower in the CYC group compared
with the control, CYC + PRP, and PRP groups (p = 0.000 for each). When the CYC + PRP group and the control and PRP groups were compared
in terms of mean oocyte count, there was no statistically significant difference between
the groups (p > 0.05). The mean number of fertilized oocytes and two-celled good quality embryos
was the lowest in the CYC group, while it was highest in the PRP only group. In the
comparison between the groups, the number of fertilized oocytes and two-celled good
quality embryos was found to be statistically significant between the CYC group and
control, CYC + PRP, and PRP groups (p = 0.009, p = 0.001, p = 0.000 for fertilized oocytes, respectively. for the number of good quality embryos;
p = 0.016, p = 0.002, p = 0.000).
Fig. 1 Comparison of the mean of metaphase I (M1), metaphase II (M2) oocytes and germinal
vesicles (GVs) in experimental groups. Abbreviations: Control, control group; CYC,
cyclophosphamide administered group; CYC + PRP, cyclophosphamide and platelet rich
plasma applied group; PRP, platelet-rich plasma applied group. * p < 0.05 compared with the control, CYC + PRP, and PRP groups. Values are given as
mean and standard error.
Fig. 2 Comparison of the mean numbers of total oocytes, fertilized oocytes, and two-celled
good quality embryos in the experimental groups. Abbreviations: Control, control group;
CYC, cyclophosphamide administered group; CYC + PRP, cyclophosphamide and PRP applied
group; PRP, platelet-rich plasma applied group. * p < 0.05 compared with control, CYC + PRP, and PRP group. Values are given as mean
and standard error.
Two-celled embryos were obtained by culturing oocytes after IVF. In the CYC group,
the quality of the two-celled embryo was very poor, a high rate of fragmentation was
seen. Although embryos with equal blastomeres were seen in the CYC + PRP and PRP groups,
embryos with a small amount of fragmentation were seen in the CYC + PRP group. This
effect was thought to be due to CYC. In the control group, embryos with equal blastomeres
were generally seen, however, it was seen in embryos with fragmentation ([Fig. 3]).
Fig. 3 Oocyte and two-cell embryo images of control, CYC, CYC + PRP and PRP groups, 200X
magnification in inverted microscope. There are two M2 oocytes belonging to the control
group, 3 M2 and 1 GV oocyte in the CYC + PRP group and 4 M2 oocytes in the PRP group.
In the CYC group, there are 2 GV oocytes whose ooplasms are severely damaged. In the
CYC group, the quality of the 2-cell embryo is very poor, with a high rate of fragmentation.
While two-cell embryos with equal blastomeres are seen in the CYC + PRP and PRP groups,
embryos with a very small amount of fragmentation are seen in the CYC + PRP group.
Although embryos with equal blastomeres were seen in the CYC + PRP and PRP groups,
embryos with a small amount of fragmentation were seen in the CYC + PRP group. Abbreviations:
Control, control group; CYC, cyclophosphamide administered group; CYC + PRP, cyclophosphamide
and platelet-rich plasma applied group; PRP, platelet-rich plasma applied group; GV,
germinal vesicle; MII, metaphase II.
When the AMH concentrations in the study groups were examined, it was found that it
was the highest in the PRP group, while it was the lowest in the CYC group ([Fig. 4]). It was observed that there was a statistically significant difference between
the CYC and CYC + PRP groups when compared with the control group (p = 0.000).
Fig. 4 Comparison of experimental groups serum anti-Mullerian hormone (AMH) concentrations.
Abbreviations: Control, control group; CYC, cyclophosphamide administered group; CYC + PRP,
cyclophosphamide and PRP applied group; PRP, platelet-rich plasma applied group. p < 0.05, compared with the control group. Values are given as mean and standard error.
Discussion
For ovarian failure, the presence of ovarian atrophy, follicle reduction, and sex
hormonal diminution are used.[23] Looking at society, ovarian failure (POI) is one of the most important diseases
that cause infertility in women and threaten women's health. The early detection and
treatment of ovarian dysfunctions continues to be an important research and clinical
area of interest in gynecology. Infertile patients with aging ovaries - sometimes
called the approaching POI, their numbers are increasing day by day and constitute
a significant proportion of patients applying for IVF/ART. Current approaches to effective
management of patients diagnosed with POI offer a wide range of options. Although
egg donation (ED) is still the most successful and final treatment for POI patients,
the vast majority of these infertile women are reluctant to consent to ED upon initial
diagnostic interview and demand alternative solutions using their own autologous eggs,
despite the low chance of success.[24] Many researchers have investigated the use of stem cell transplantation, including
human menstrual blood stem cells, fat-derived stem cells, human endometrial mesenchymal
stem cells, Platelet-rich plasma (PRP), as a cell therapy to reverse ovarian damage
caused by chemotherapy.[12]
[16]
[25]
[26] PRP has been defined as a blood plasma fraction with a platelet concentration 4
to 5 times higher than the normal level, and its beneficial effect on tissue regeneration,
angiogenesis activation, inflammation control and anabolism has already been demonstrated
in many medical fields.[27] The main components of PRP that contribute to tissue healing and regeneration, anabolism
increase, differentiation and proliferation, angiogenesis activation, inflammation
control can be listed as hormones, macrophages, neutrophils, cytokines and various
growth factors.[28]
[29] Therefore, the use of PRP is considered a justified and potentially successful opportunity
to increase the fertility outcome in POI patients where the main problem is ovarian
failure.
A POI model induced by cyclophosphamide (CYC) was used in the present study. Cyc is
an alkylating agent which induce ovarian failure in animal models.[30] It has been shown in previous studies that CYC disrupts the ultrastructure of granulosa
cells and induces apoptosis and autophagy and eventually causes ovarian failure.[30]
[31] Cyc has been shown to reduce ovarian weight and volume, reduce the number of different
follicles and sex hormone levels, and increase atretic follicles.[12] In their study of agents that prevent chemotherapy-induced ovarian damage, Roness
et al.[32] noted that AS-101, AMH, imatinib, sphingosine-1-phosphate, granulocyte colony stimulating
factor, bortezomib, and multi-drug resistance gene-1 were effective in preventing
chemotherapy-induced ovarian damage.[32] Different mechanisms of action associated with different protective agents have
been shown to be effective, including inhibition of follicle activation, anti-apoptosis
effects, vascular effects, and gene upregulation.[32] When this protective effect is evaluated in terms of PRP, there are studies showing
the success of PRP.[12]
[16] These studies were generally performed on ovarian tissue and were performed on oocytes
obtained at the stage of folliculogenesis.
Growth factors play an important role in improving the structure and function of the
ovaries, and different growth factors such as VEGF, EGF, PDGF, and TGF-b have been
shown to have protective effects on ovarian damage.[13]
[14]
[33] Platelet-rich plasma has a protective effect against ovarian damage caused by CYC,
as it has high amounts of these factors in its structure. This efficiency has been
demonstrated in previous studies.[12]
[16] This protective feature of PRP in POI patients is to protect follicle development
and oocyte number during folliculogenesis. Except for the protective effects of PRP
on the ovary, there are many studies on the effects of PRP on the endometrium.[34] It has been shown that intrauterine PRP treatment supports endometrial growth and
improves assisted reproductive outcome in patients with thin endometrium.[35] In humans, PRP used in autologous ovarian transplantation to improve the vascularization
and quality of the implant has been shown to increase transplant success resulting
in live birth.[36] There is no study on the effectiveness of PRP for POI patients who have serious
difficulties in IVF applications. Human studies on the subject in the literature are
only at the level of case reports.[37]
[38]
As a result of our study, it is seen that the addition of PRP treatment in the group
where POI was created with CYC positively affected the results of subsequent IVF.
This positive effect is valid for both the number of oocytes obtained by ovulation
stimulation and the number of embryos on day 2 obtained after fertilization. When
the day-2 embryos obtained were evaluated in terms of their quality, it was noted
that there is a significant difference in the PRP applied group compared with the
untreated group. This may be due to the fact that the PRP treatment could probably
enrich the dysfunctional ovarian tissues with essential factors for neoangiogenesis,
leading to tissue regeneration and reactivation. Although the effect of PRP on regenerative
and repair processes in somatic tissues remains largely uncertain, growth factors
contained in PRP content may have many critical roles in the ovaries through physiologically
local effects such as cell growth, proliferation, differentiation, chemotaxis, angiogenesis,
and formation. These growth factors control the release of the extracellular matrix
and even other growth factors in close proximity to the release sites.[39] Platelet-rich plasma can accelerate this process while supporting the self-repair
of ovaries, follicles after chemotherapy, which already have the potential to repair
itself.[40]
When the control group and the PRP-only group were compared, the number of oocytes
obtained in the group receiving PRP and the number of embryos on day 2 were higher,
but this result was not found to be statistically significant. This situation makes
us think that PRP does not have a significant effect in conditions with normal ovarian
function and reserve. It seems, the beneficial effect of PRP is only applied on damaged
ovaries, and it has no effect on the normal structure for IVF cycles.
Anti-Mullerian hormone, a powerful marker of ovarian reserve, is a member of the transforming
growth factor superfamily produced by the granulosa cells of the antral follicles
in the ovary.[41] Considering the AMH levels, there was an increase in the PRP-CYC group compared
with the CYC group. However, the AMH levels following PRP treatment corresponded to
the expected lower AMH levels in a POI case, although an improvement in overall reproductive
potential was observed. Although the PRP-CYC group had a low AMH level that could
be diagnosed with POI compared with the control group, this decrease is not as extreme
as in the group without PRP treatment, and it is not at a low level that will allow
more oocytes and embryos to be obtained as a result of IVF. This suggested that PRP
could improve ovarian reserve by protecting ovarian granulosa cells. This evidence
demonstrated the protective effects of PRP from CYC damage to the ovarian follicles.
Conclusion
The present study evaluated the number and quality of oocytes obtained after ovarian
stimulation and the number and quality of embryos obtained on the second day after
fertilization. Our study showed that PRP can protect the ovarian function against
damage induced by CYC, but it provides an improvement in the number of oocytes and
developing embryos as a result of the oocyte stimulation performed during the subsequent
IVF procedure. However, investigating the implantation results of these embryos, and
evaluating the ongoing pregnancy results will be a good target for future studies.
