FACOLTA’ DI MEDICINA E CHIRURGIA
Dipartimento di Medicina della Procreazione e dell’Età Evolutiva
Divisione di Ginecologia ed Ostetricia “P. Fioretti”
Direttore: Prof. A.R. Genazzani
TESI
DI
DOTTORATO
DI
RICERCA
“Correlation between angiogenic growth factors and
perifollicular blood flow in patients undergoing in
vitro fertilization”
Relatore:
Chiar.mo Prof. A.R. Genazzani
Candidato:
Patrizia Monteleone
A
BSTRACTWhile primordial and primary follicles receive nutrients and oxygen by passive diffusion from stromal blood vessels, follicular growth is associated with the development of an individual capillary network and continued angiogenesis to nourish the rapidly expanding follicle. It seems, however, that age is negatively correlated with ovarian perifollicular blood flow even if this has been observed only very late in the follicular phase. Support to this finding is the observation of increased levels of Vascular Endothelial Growth Factor (VEGF) in the follicular fluid from aging women. Transcriptional upregulation of VEGF is involved in the cellular adaptation to hypoxia under control of Hypoxia-Inducible Factor 1 (HIF1), a transcription factor activated by low oxygen tension to prevent depletion of oxygen at anoxic levels and subsequent cell death. Various recent studies have discovered that the vascular and the nervous system share an overlapping repertoire of growth factors affecting the development and the homeostasis of both systems. These include members of the vascular endothelial growth factor (VEGF), and neurotrophin growth factor family, such as brain derived neurotrophic factor. It has been shown, in neural tissue, that BDNF activation of TrkB stimulates
tissues, BDNF seems to act as a proangiogenicstimulus.In the ovary, both VEGF and BDNF, secreted by the granulosa cells, seem to play a role in folliculogenesis and oocyte maturation. We aimed at establishing whether there is a relationship between follicular fluid VEGF concentrations, BDNF concentrations and perifollicular blood flow (PFBF) in women undergoing IVF and whether age makes a difference. In a prospective observational study, we enrolled 30 consecutive patients all at their first IVF cycle. These were subdivided in two age groups (13 patients aged 30-34 years and 17 patients aged 35-39 years). At oocyte retrieval, the perifollicular vascularity of one follicle per ovary was estimated qualitatively through power Doppler blood flow, for a total of 60 follicles. The follicular fluid from each of the identified follicles was centrifuged and stored until VEGF and BDNF assay. In our study, we found VEGF levels to be significantly correlated with grade of PFBF only in the younger age group. BDNF did not directly correlate with PFBF in any of the patients. There was a significant positive correlation between VEGF and BDNF follicular fluid levels only in the younger patients. VEGF follicular fluid levels were significantly higher in the older age group than in the younger group (VEGF 955,4 vs 546,5 ng/ml);. BDNF levels were also significantly higher in the older versus younger women undergoing
IVF (BDNF 603,3 ng/ml vs 401,1 ng/ml). The ability of a given follicle to express BDNF and VEGF and develop an adequate vascular network may be inter-related. An adequate blood supply is of fundamental importance in the regulation of intrafollicular oxygen levels and the determination of oocyte quality. Reproductive aging may be associated with relatively low levels of intrafollicular oxygen and with an attempt, on behalf of a given follicle, to increase its vascular supply by increasing the secretion of VEGF. The trigger for this phenomenon may be represented by BDNF, which is tightly correlated to VEGF levels in younger age women, and tends to increase in older women.
INDEX
I. INTRODUCTION……….………….P.1
A. THE IMPORTANCE OF ANGIOGENESIS DURING OVARIAN
FOLLICULOGENESIS…..………...P.1
B. PERIFOLLICULAR BLOOD FLOW AND REPRODUCTIVE
OUTCOME……….P.2
C. AGING AND PERIFOLLICULAR BLOOD
FLOW……….………..P.3
D. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND
BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF): A COMMON
GOAL………..……….P.4
E. AGING AND FOLLICULAR EXPRESSION OF
VEGF……...……….…P.6 II.AIM………P.8
III.MATERIALS AND METHODS………..………...…….P.9
A. SUBJECTS……….…………...P.9
B. TREATMENT PROTOCOL….………..……..……….P.10
C. EVALUATION OF PERIFOLLICULAR BLOOD FLOW….…….P.10
D. VEGF ASSAY……....…….……….………...P.13
E. BDNF ASSAY….………………...……….………....P.13
IV. STATISTICAL ANALYSIS………...…….………P.14
V. RESULTS……….………….…….………P.15
VI. DISCUSSION……….….….………P.17
VII. REFERENCES……..……….………..P.22
I.
Introduction
A. THE IMPORTANCE OF ANGIOGENESIS DURING OVARIAN
FOLLICULOGENESIS
Aside from wound healing and certain pathological processes, including neoplasia, the vascular system in the adult is generally quiescent. An exception takes place in the ovaries where there is intense angiogenesis and increased permeability of blood vessels during follicular development, ovulation and subsequent formation of the corpus luteum. Furthermore, angiogenesis is regulated independently within each individual follicle and depending on the extent of the vascular plexus and permeability of vessels, the supply of large molecular weight tropic factors, precursors and lipids can be controlled. This indicates the follicular vasculature could be intimately involved in the processes of follicular selection, dominance and atresia. It is likely that some types of infertility are associated with a disturbance of follicular angiogenesis resulting in inadequate development. Indeed, while primordial and primary follicles receive nutrients and oxygen by passive diffusion from stromal blood vessels, follicular growth is associated with the development of an individual capillary network and continued angiogenesis to nourish the rapidly expanding follicle. The vascular
sheath that develops around each follicle is confined to the thecal layer by the presence of the membrana propria until the breakdown of the basement membrane at ovulation.
B. PERIFOLLICULAR BLOOD FLOW AND REPRODUCTIVE
OUTCOME
The quality of an oocyte is the chief determinant of embryo quality. It has become increasingly clear that the follicular microenvironment of a human oocyte is a crucial factor for its
developmental competence (1).
Indeed the quality and maturity of an oocyte is influenced by the intrafollicular level of oxygen content which, in turn, is proportional to the degree of follicular vascularity (1). The regulation of follicle growth also depends on the availability of an adequate vascular supply to provide nutrients as well as regulatory signals (2). In contrast to primordial and preantral follicles which derive their blood supply from the stromal vessels, growing follicles clearly depends on a sufficient ingrowth of capillaries into the theca (3). It is well known that dominant follicles have not only a more vascular theca, but also an increased uptake of serum gonadotrophins compared with other follicles (2). In a prospective study based on pulsed Doppler ultrasonographic analysis,
Huey et al. (4) found that oocytes deriving from follicles with optimal vascularization and oxygen content (≥3%) had higher fertilization and
developmental potential. Furthermore, studies of perifollicular
vascularity before oocyte aspiration by transvaginal power Doppler ultrasonography, which enables a sensitive analysis of the microvessels surrounding each follicle, reported a positive correlation between high grade vascularity and improved outcome during IVF cycles (5, 6).
C. AGING AND PERIFOLLICULAR BLOOD FLOW
It was suggested that an important environmental factor responsible for oocyte senescence might be represented by a reduced oxygen supply to the leading follicle, a condition dependent on a compromised perifollicular vascularization (3, 7). Indeed, modifications found in old MII oocytes, such as spindle and chromosome abnormalities, were reported to resemble those occurring in young oocytes obtained from Graafian follicles with reduced perifollicular vascularization and oxygen content (1, 7). To our best knowledge, however, the question as to whether advancing age is associated with decreasing ovarian follicular blood flow has been so far poorly investigated. Recently, Costello et al. (8) described for the first time a significant negative correlation between
age and ovarian perifollicular blood flow which was only observed very late in the follicular phase of ovarian stimulation.
D. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND
BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF): A COMMON
GOAL
Development and homeostasis of the vascular and nervous system were believed to be controlled by different, specialized growth factors and their receptors. Yet various recent studies have discovered that the vascular and the nervous system share an overlapping repertoire of growth factors affecting the development and the homeostasis of both systems. This suggests the evolution of universally applicable molecular mechanisms to control path finding, spatial patterning, proliferation and protection. In recent years, the number of shared growth factors discovered to be important for both systems increases continuously. One prominent example for a shared growth factor is VEGF, an important stimulator of blood vessel growth during development and in the adult. VEGF induces angiogenesis, the generation of new blood vessels from pre-existing vessels, then results in the further expansion of the primary vascular plexus (9). Another such factor is BDNF. The presence of
BDNF in the follicular fluid of the human ovarian follicle, its secretion by human cumulus granulosa cells and its promotion of mouse oocyte maturation was first reported by Seifer et al. in 2002 (10). The fact that Trk B receptor, principal target of BDNF action, was identified in preovulatory human unfertilized oocytes suggests that BDNF may have a role in supporting granulosa cell-oocyte communication within the follicle (11). Indeed BDNF seems to mediate LH and HCG actions in promoting preovulatory oocyte meiotic maturation (12) In vitro mouse studies have provided a direct demonstration that BDNF has a role in the maturation of the oocyte, promoting extrusion of first polar body (10).
In neuroblastoma cells, BDNF activation of TrkB has been shown to stimulate VEGF mRNA transcription via induction of HIF-1 (13). This increase in VEGF mRNA leads to an increase in VEGF secreted by
neuroblastoma cells. Moreover, BDNF seems to induce VEGF
expression via the TrkB/PI3K/mTORpathway. BDNF induction of
HIF-1 is a major mediator of the increased expression of VEGF in TrkB-expressing neuroblastoma cells. BDNF stimulates an increase in HIF-1 expression that increases VEGF transcription leading to an increase in VEGF secretion by neuroblastoma cells. Thisis similar to the effects of BDNF on normal neural tissues. This study identifies a potential mechanism for neurotrophin regulation of VEGF expression in neural-derived cells by finding that BDNF induction of HIF-1 mediates the
increase in VEGF transcription. Recent studies show that hypoxia induces BDNF (14), which has a protective effect on normal neural tissue that may be related to vascular adjustments (14). Whether the
increase in BDNF underhypoxic conditions requires induction of HIF-1
or whether BDNF-stimulated increases in HIF-1 are required for
neuroprotection against hypoxia in normal tissues remains to be
elucidated. BDNF activation of TrkB promotes angiogenesis in the developing embryonic myocardium (15), recruits brain-derived (16) and bone marrow-derived TrkB expressing endothelial precursor cells from the bone marrow (14) and increases VEGFR on endothelial cells (16). Thus, in normal tissues, neurotrophins act as proangiogenic stimuli
E. AGING AND FOLLICULAR EXPRESSION OF VEGF
It has been observed that increased levels of VEGF are present in the follicular fluid from aging women (17, 18). Transcriptional upregulation of VEGF is involved in the cellular adaptation to hypoxia under control of HIF1 (Hypoxia-Inducible Factor 1), a transcription factor activated by low oxygen tension (19) to prevent depletion of oxygen at anoxic levels and subsequent cell death (20, 21). This growth factor plays a central role in the regulation of angiogenetic processes in
the ovary (22) and in the growth of the ovarian follicle (23), where granulosa and theca cells are the main producers of VEGF in response to gonadotrophin (24). Although the cause for potential age-related decline in ovarian follicle vascularity remains unknown, the presence of elevated levels of VEGF along with reduced blood flow in the follicular environment of aged ovaries suggests that, in an attempt to compensate for hypoxia, granulosa and theca cells increase the synthesis of VEGF which nevertheless fails in completing the adaptive response. This could be ascribed to a low responsiveness of endothelial cells consequent to possible defective signalling pathways or to an increased distance between the perifollicular bed and the wall of the growing follicle in
II. A
IMOn the basis of these findings, we aimed at establishing whether there is a relationship between follicular fluid VEGF and BDNF concentrations and whether these angiogenic factors are correlated to perifollicular vascularity and reproductive outcome in normal responder patients belonging to different age groups undergoing IVF.
III.
M
ATERIALS ANDM
ETHODSThe present study was approved by the Ethical Committee of the University of Pisa and was carried out according to rules of good clinical practice. Informed consent was obtained from each patient.
A. SUBJECTS
In a prospective observational study, we enrolled twenty-nine consecutive patients between January 2006 and January 2007 at the Centre of Reproductive Pathophysiology and Assisted Reproduction of the Pisa University Hospital. All patients were at their first IVF or IVF with intracytoplasmic sperm injection (ICSI) and embryo transfer cycle. All patients had primary infertility due to male factor or tubal factor. Only normal responders to controlled ovarian hyperstimulation, i.e. presenting a number of follicles ≥ 3. Patients with endometriosis or polycystic ovary syndrome were excluded because it is well known that these patients have higher intrafollicular VEGF levels than other infertile patients (25, 26). Morover only patients with a baseline Day 3 FSH level of < 10 UI/mL were included in the study.
The patients were subdivided in two age groups:
Group A having and age range between 30 and 34 years Group B having an age range between 35 and 39 years
B. TREATMENT PROTOCOL
Controlled ovarian stimulation was carried out with 2 to 6 ampoules/day, according to basal FSH levels and age, of recombinant FSH (Gonal F®, Serono, Italy) after a pre-treatment with oral contraceptives. All patients were administered cetrorelix (Cetrotide®, Serono, Italy), a GnRH antagonist, according to a personalized regimen, i.e. when the lead follicle reached 14 mm in diameter, to prevent premature ovulation. Recombinant HCG (Ovitrelle®, Serono, Italy) was administered when at least 2 follicles reached a mean diameter of 18 mm. After approximately 36 hours, transvaginal follicular aspiration was performed for oocyte retrieval.
The follicular fluid from the studied follicles was centrifuged and stored at -20°C until VEGF assay. The maturity and eventual fertilization of the individual corresponding oocytes were recorded as well as the quality of the embryos deriving from those oocytes according to the method described by Veeck LL (27) and pregnancy rates in women with
a given perifollicular blood flow grading. IVF or IVF-ICSI and embryo transfer were performed as appropriate.
C. EVALUATION OF PERIFOLLICULAR BLOOD FLOW
The perifollicular vascularity of one follicle per ovary per patient was estimated immediately prior to oocyte retrieval through power Doppler blood flow analysis (GE medical system Logic 3 con power Doppler). Follicles were graded according to the percentage of follicular circumference in which most flow was identified from a single cross-sectional slice as described by Chui DKC et al (28). The grading system was as follows: < 25 % follicular circumference in which blood flow was identified (F1), 26-50% (F2), 51-75% (F3), 76-100% (F4).
F1: ≤ 25% vascularized perifollicular circumference
F3: 51% - 75% vascularized perifollicular circumference
D. VEGF ASSAY
VEGF follicular fluid levels were measured by ELISA (Endogen® Human VEGF ELISA Kit, Pierce Biotechnology, Inc., Rockford). Sensitivity was < 8.0 pg/ml, intraassay and interassay coefficients of variation were 8.9% and 9.8%, respectively.
E. BDNF ASSAY
Plasma levels of BDNF were determined with a previously described (29) enzyme-linked immunosorbent assay (ELISA) method (BDNF Emax Immunoassay System, Promega, USA), after appropriate dilution of samples (1:4) using Block & Sample Buffer, according to manufacturer instructions. The sensitivity of the assay, expressed as a minimal amount of BDNF distinguishable from the zero sample with 95% probability was 15.6 pg/ml, and the intra- and inter-assay coefficients of variation were 6.0% and 8.5%, respectively. The antiserum employed was highly specific, showing less than 3% cross-reactivity with other related neurotrophic factors (nerve growth factor, NT-3 and NT-4) at 100 ng/ml, as specified by the manufacturer.
IV.
S
TATISTICAL ANALYSISResults are expressed as mean ± SD. Between-group differences were evaluated by means of Student T-test. The correlation between perifollicular vascularity and VEGF and BDNF levels was verified by means of Pearson's method. A p value of <0.05 was considered statistically significant.
V.
R
ESULTSA total of 60 follicles were studied in 30 patients. Of the 30 patients, 13 were aged between 30 and 34 years (Group A) and 17 were aged between 35 and 39 years (Group B). Of the 26 evaluated follicles in Group A, none were graded as F1, 12 were graded as F2, 4 were graded as F3 and 10 were graded as F4. Of the 34 follicles evaluated in Group B, 19 were graded as F1, 11 as F2, 4 as F3 and none as F4. There was no significant difference in follicular size.
In our study, we found VEGF levels to be significantly correlated with grade of perifollicular blood flow (PFBF) only in Group A (Pearson correlation index 0.86), while BDNF levels did not correlate with PFBF in either group. BDNF and VEGF levels were tightly correlated in Group A patients (Pearson correlation index 0.84). However, this correlation was lost in Group B patients, i.e. older than 35 years.
There was a significant difference in VEGF levels and in BDNF levels between age groups, that is VEGF and BDNF levels were significantly higher in the older patients, although the increase in VEGF levels was much greater (VEGF 955,4 ± 423,6 in group B patients vs 546,5 ± 587,9 ng/ml in group A patients; BDNF 603,3 ± 342,6 in group B patients vs 401,1 ± 138,6 pg/ml in group A patients) (Figure 1 (a)).
Perifollicular vascularity on the day of oocyte retrieval was significantly higher in the younger patients, i.e. with a mean higher percentage of perifollicular circumference showing a power Doppler signal (72 ± 23% in Group A versus 44 ± 18% in Group B) (Figure 1 (b))..
Statistical analysis revealed no difference in ICSI percentage rate between the two age groups. Oocytes obtained from follicles of Group A patients showed a higher grade of fecundibility than those obtained from Group B patients (95,6% fertilization rate in group A patients vs 72,7% fertilization rate in group B patients). Moreover, the percentage of grade A embryos was greater in the younger patients (44,0% in Group A vs 22,2% in Group B). No triploid embryos were recorded. Finally, as expected, the pregnancy rate per embryo transfer was higher in women aged less than 34 years (46,2%) with respect to those aged over 35 years (17,6%)
VI.
D
ISCUSSIONIt is known that the most common factors responsible for implantation failure of embryos in patients undergoing IVF are oocyte cytoplasmic or chromosomal defects (3). Oocytes are very sensitive to hypoxic damage (1). A sufficient supply of oxygen seems to be mandatory for the formation and stability of the meiotic spindle and the development of a given embryo (1). In light of the fact that the oocyte depends on the diffusion of oxygen particles from the thecal microvasculature through the follicular basement membrane granulosa cells, follicular fluid and zona pellucida, the ability of a given follicle to develop an adequate vascular network is crucial. An adequate blood supply is of fundamental importance in the regulation of intrafollicular oxygen levels, provision of growth factors, nutritional factors and gonadotropins and the determination of oocyte quality.
Chui and Bhal have demonstrated a strong association between development of perifollicular vascularity and ability of an embryo to implant (28, 30). Costello et al., however, recently reported that age is negatively correlated with ovarian perifollicular blood flow even if this has been observed only very late in the follicular phase (8). Support to this finding is the observation of increased levels of VEGF
in the follicular fluid from aging women (17, 18). Transcriptional upregulation of VEGF is involved in the cellular adaptation to hypoxia under control of Hypoxia-Inducible Factor 1 (HIF1), a transcription factor activated by low oxygen tension to prevent depletion of oxygen at anoxic levels and subsequent cell death. It has been shown, in neural tissue, that VEGF mRNA transcription via induction of HIF-1 is induced by BDNF activation of its receptor TrkB. Indeed, in neural
tissues, BDNF seems to act as a proangiogenicstimulus.In the ovary,
both VEGF and BDNF, secreted by the granulosa cells, seem to play a role in folliculogenesis and oocyte maturation. In particular, during folliculogenesis, VEGF secretion, which is induced by gonadotropins, determines the formation of a vascular network in the thecal cell layer of the follicle (24, 31). BDNF, on the other hand, seems to be involved in in supporting granulosa cell-oocyte communication within the follicle (11) and in promoting the maturation of the oocyte, promoting extrusion of first polar body (10).
In the present study, we aimed at establishing whether there is a relationship between follicular fluid VEGF and BDNF concentrations and whether these angiogenic factors are correlated to perifollicular blood flow (PFBF) and reproductive outcome in normal responder patients undergoing IVF. Moreover, the effect of age on these
parameters was evaluated by comparing women aged between 30 and 34 years (Group A) and women aged between 35 and 39 years (Group B).
We found VEGF levels to be significantly correlated with grade of PFBF in the younger age group of patients. This is a plausible finding because VEGF is directly involved in the perifollicular angiogenesis that takes place at each ovulatory cycle (24) and therefore the amount by which it is secreted most probably directly correlates to the extent of the perifollicular vascular bed (32). However, in the older patients, this correlation was lost as the increase in VEGF levels (VEGF 955,4 in Group B vs 546,5 ng/ml in Group A), that confirm data already present in the literature (17,18), was actually associated to a general reduction in perifollicular vascularity (72% vascularised follicular circumference in Group A versus 46% vascularised follicular circumference in Group B). The reason why this increase in VEGF concentrations does not lead to a successful compensatory increase in perifollicular blood flow in the older women is not clear. Further studies should be performed to analyse the concentration of the soluble VEGF receptor to measure the actual VEGF bioavailability. Increased VEGF levels in patients undergoing IVF have been associated to bad embryo quality in the past medical
literature (33). Indeed the percentage of Grade A embryos in our casistic of older patients was much lower than that found in the younger patients (44,0% in Group A vs 22,2% in Group B). As expected, the pregnancy rate per embryo transfer was higher in women aged less than 34 years (46,2%) with respect to those aged over 35 years (17,6%)
BDNF did not directly correlate with PFBF in any of the patients. However, it is also true that BDNF is not a direct angiogenic factor; it must act through VEGF (13). In the younger patients, follicular fluid BDNF levels were found to correlate positively with VEGF levels. Moreover, as for VEGF, follicular fluid BDNF levels were significantly higher in the older versus younger women undergoing IVF (BDNF 603,3 ng/ml vs 401,1 ng/ml). This finding is quite interesting in our opinion because it may suggest an actual modulating role on behalf this neurotrophin on perifollicular angiogenesis through the increase in VEGF synthesis (13), both in younger patients and even more so in the older patients, where the
presence of hypoxia may stimulate VEGF mRNA transcription via
induction of HIF-1 (13).
In conclusion, the secretion of BDNF and VEGF levels in follicular fluid seem to be strongly correlated in patients under the age
of 34. In younger women, the ability of a given follicle to express BDNF and VEGF and develop an adequate vascular network may be inter-related. Aging is associated with an increase in follicular fluid BDNF and VEGF levels but with a decrease in perifollicular vascularity. Reproductive aging may be associated with relatively low levels of intrafollicular oxygen and with an attempt, on behalf of a given follicle, to increase its vascular supply by increasing the secretion of VEGF. The trigger for this phenomenon may be represented by BDNF, which is tightly correlated to VEGF levels in younger age women, and tends to increase in older women. The reason why the increase in angiogenic factors is not associated to a compensatory increase in perifollicular blood flow remains to be elucidated and requires further studies to elaborate therapeutical strategies that may help improve reproductive outcome in older patients.
VII.
R
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