Gynecological Endocrinology, April 2008; 24(4): 184–187
ASSISTED REPRODUCTIVE TECHNOLOGY
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Vascular endothelial growth factor level changes during human embryo development in culture medium
PAOLO GIOVANNI ARTINI, VALERIA VALENTINO, PATRIZIA MONTELEONE, GIOVANNA SIMI, MARIA ROSARIA PARISEN-TOLDIN, FRANCESCA CRISTELLO, VITO CELA, & ANDREA RICCARDO GENAZZANI Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Pisa, Italy (Received 27 November 2007; accepted 21 December 2007)
Abstract Objective. Implantation is a complex phenomenon consisting of the first strong contact between embryo and endometrium. Recent studies have demonstrated that this process is dependent not only on the ‘readiness’ of the endometrium, but also on complex interactions between endometrial and embryonic tissues that cross-talk by means of different molecules (growth factors, cytokines, vasoactive factors). Investigations performed on human blastocysts indicate a role for vascular endothelial growth factor (VEGF) in these processes. The aim of the present study was to investigate VEGF levels at different stages in human embryo culture medium. Study design. We selected 20 women among patients undergoing assisted reproduction with the in vitro fertilization– blastocyst transfer protocol. The oocytes were inseminated by intracytoplasmic sperm injection. For each patient, approximately two cultures of four microinjected oocytes (and then of four embryos) were performed. Each culture of four oocytes/ embryos was placed in one dish to increase the probability to detect small VEGF concentrations. Results. Results showed significantly higher VEGF levels in the medium at blastocyst stage (12.16 + 2.80 pg/ml) compared with embryos at pronuclear stage (13.58 + 2.32 pg/ml) and microinjected oocytes (12.80 + 3.45 pg/ml). Conclusions. An important VEGF synthesis by blastocysts occurs during human embryo development.
Keywords: Angiogenesis, implantation, in vitro fertilization, vascular endothelial growth factor
Introduction Implantation is the result of interaction between the embryo and the endometrium, a process that begins around the 7th day after fertilization. Implantation consists of three main phases: embryo apposition and attachment to the maternal endometrial epithelium, traversing adjacent cells of the epithelial lining and invasion into the endometrial stroma. The invasive phase is characterized by attachment of the invading trophoblast to the extracellular matrix, degradation of the matrix and replacement of the decidual artery endothelium with a new trophoblast phenotype called the vascular trophoblast [1]. Recent studies have demonstrated that this process is dependent not only on the ‘readiness’ of the endometrium, but also on complex interactions between endometrial and embryonic tissues that cross-talk by means of different
molecules (growth factors, cytokines, vasoactive factors) [2–4]. Vascular endothelial growth factor (VEGF) is an approximately 46-kDa dimeric glycoprotein [5,6] that plays a key role in angiogenic processes. The synthesis of the growth factor occurs predominantly in endothelial cells, macrophages and granulosa cells. Human chorionic gonadotropin (hCG), which has an important role in early pregnancy, is capable of inducing VEGF synthesis in vitro and in vivo [7,8]. VEGF mediates neovascularization in various biological processes, such as implantation, the menstrual cycle, corpus luteum development, ovarian follicle development, embryogenesis and tumorigenesis [9]. It may also be a factor responsible for maintaining perifollicular blood flow and regulation of intrafollicular oxygen levels [10].
Correspondence: P. G. Artini, Division of Obstetrics and Gynecology, University of Pisa, Via Roma 35, I-56100 Pisa, Italy. Tel: 39 50 992223. Fax: 39 50 553410. E-mail:
[email protected] ISSN 0951-3590 print/ISSN 1473-0766 online ª 2008 Informa UK Ltd. DOI: 10.1080/09513590801893117
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VEGF levels in human embryo culture medium A study in an animal model has indicated that VEGF-A164 is the isoform of the growth factor most expressed in endometrial tissue from the first day of pregnancy. It has also suggested that NRP1 (neuropilin-1) receptor could increase the binding between VEGF and its specific receptor [11]. Further animal studies revealed that VEGF and its Flt-1 receptor are greatly synthesized by deciduas and the embryo beginning on the 4th day after fertilization; in these first phases, vascular endothelium is not able to produce the growth factor [12]. A study performed, for the first time, on human blastocysts demonstrated the presence of VEGF mRNA. Furthermore, VEGF mRNA splice variants were differentially expressed in human blastocysts: VEGF145 is the isoform of the growth factor most secreted by the embryo [13], and it is the isoform that we search in our culture media. The aim of the present study was to investigate VEGF levels in human embryo culture medium.
Materials and methods Patients and protocol We selected 20 women among patients undergoing assisted reproduction with the in vitro fertilization (IVF)–blastocyst transfer protocol; selection criteria were age 535 years and approximately 15 oocytes collected on the day of the oocyte retrieval. The cause of IVF treatment was male infertility and the chosen IVF technique was intracytoplasmic sperm injection (ICSI), to obtain higher fertility rate. All patients underwent pituitary downregulation by means of intramuscular administration of a gonadotropin-releasing hormone analog, triptorelin 3.75 mg (Decapeptyl1; Ipsen, Italy), in the early follicular phase (day 1 to day 3 of the menstrual cycle); the downregulation effect was verified in each patient after 10–14 days. When the suppressive effects (estradiol 550 pg/ml, no cysts or ultrasound follicles 41.0 cm in maximum diameter) were obtained, controlled ovarian hyperstimulation was begun by administering 150–300 IU of recombinant follicle-stimulating hormone (Gonal-F1; Serono, Italy) (75 IU/ampoule). On day 6 of the cycle, we started to evaluate follicular growth by transvaginal ultrasound examination using a 6.5-MHz vaginal transducer (Ansaldo AU590 Asynchronous; Genova, Italy) and plasma estradiol levels were assessed daily by radioimmunoassay (Radim; Pomezia, Italy). When at least one follicle of 16 mm maximum diameter and plasma estradiol levels of at least 200 pg/ml per follicle were present, 10 000 IU of hCG (Profasi1; Serono, Italy) were administered. Oocyte retrieval was performed by transvaginal ultrasound-guided follicle aspiration 34–38 h after hCG administration. Table I shows the principal
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Table I. Principal characteristics of the women studied and the intracytoplasmic sperm injection procedure. Patients’ age (years) Infertility duration (years) Serum E2 at day of pick-up (pg/ml) No. of FSH ampoules Stimulation days at pick-up (n) No. of aspirated follicles No. of recruited oocytes
30.2 + 3.5 4.2 + 1.8 1255.5 + 575.2 36.5 + 17.3 14.2 + 1.14 20.4 + 3.2 17.8 + 1.5
E2, estradiol; FSH, follicle-stimulating hormone; data are expressed as mean + standard deviation.
characteristics of the women studied and of the ICSI procedure. Embryo culture The retrieved oocytes were scored for maturity on the basis of low, medium or high cumulus expansion. The oocytes were inseminated by ICSI to obtain higher fertility rate. For each patient, approximately two cultures of four microinjected oocytes were carried out (time 0– 24 h) in HTF (Human Tubal Fluid) medium (Irvine Scientific, Santa Ana, CA, USA); we placed four microinjected oocytes in one dish to increase the probability of detecting small VEGF concentrations. After 24 h the HTF medium of every dish was collected and stored at 7208C until evaluation; the pronuclear embryos obtained were transferred to dishes (groups of four pronuclear embryos to increase the possibility of detecting small VEGF concentrations) containing fresh P1 (Preimplantation stage 1) medium (Irvine Scientific). After 24 h the P1 medium of every dish was collected and stored at 7208C until evaluation; the 2–8-cell embryos obtained were transferred to dishes (groups of four embryos) containing fresh P1 medium. After 24 h the P1 medium of every dish was collected and stored at 7208C until evaluation; the morulas obtained were transferred to dishes (groups of four morulas) containing Blastocyst medium (Irvine Scientific). After 24 h the Blastocyst medium of every dish was collected and stored at 7208C until evaluation; the blastocysts obtained were transferred to dishes (groups of four blastocysts) containing Blastocyst medium. After 24 h the Blastocyst medium was collected and stored at 7208C until evaluation; the blastocysts obtained were transferred in utero or were frozen. With this study design, for every step in the culture procedure we were able to obtain samples of medium from dishes in which the four oocytes/embryos were at the same development stage (microinjected oocytes, pronuclear embryos, 2–8-cell embryos, morulas, blastocysts).
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The use of sequential culture media (HTF, P1 and Blastocyst) guarantees the best culture conditions to obtain the greatest number of blastocysts, according to many authors [16]. VEGF levels in the culture medium were detected by enzyme immunoassay (VEGF Human, Biotrak; Amersham Biosciences, Switzerland) having a sensitivity 58.0 pg/ml and range of 3.13–2000 pg/ml; intra-assay error is +4.2% and inter-assay error is +6.3%.
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Statistical analysis Comparisons between VEGF levels in culture media from the different dishes were made by the t test. Results are expressed as mean + standard deviation. A probability p 5 0.05 was considered as statistically significant. Results Results are presented in Table II. VEGF levels were first determined in the three culture media used (HTF, P1 and Blastocyst); we found no differences in mean VEGF concentrations. Medium from embryos at pronuclear stage (164 embryos) showed VEGF levels similar to those of microinjected oocytes (204 oocytes) (13.58 + 2.32 pg/ml vs. 12.80 + 3.45 pg/ml). Medium from 2–8-cell embryos (136 embryos) presented higher VEGF levels (16.07 + 2.71 pg/ml), but the difference was not statistically significant. Medium from morulas (108 embryos) showed VEGF levels similar to those of 2–8-cell embryos (136 embryos) (16.28 + 3.98 pg/ml vs. 16.07 + 2.71 pg/ml). BlasTable II. Vascular endothelial growth factor (VEGF) levels measured in culture media.
Medium Fertilization medium Cleavage medium Blastocyst medium Microinjected oocytes Pronuclear stage embryos 2–8-cell embryos Morulas Blastocysts
Time after No. of microinjection No. of oocytes/ (h) dishes embryos
VEGF (pg/ml)
–
8
–
11.78 + 3.79
–
8
–
10.55 + 3.01
–
8
–
12.16 + 2.80
24
51
204
12.80 + 3.45
48
41
164
13.58 + 2.32
72
34
136
16.07 + 2.71
96 120
27 23
108 92
16.28 + 3.98 21.05 + 2.12*
VEGF levels are expressed as mean + standard deviation; *significant difference vs. pronuclear stage and microinjected oocytes: p 5 0.05.
tocysts medium (92 embryos) showed significantly higher VEGF levels (21.05 + 2.12 pg/ml) compared with medium from embryos at pronuclear stage (p 5 0.05) and medium from microinjected oocytes (p 5 0.05). Discussion The present study is the first report of dosable VEGF levels in embryo culture medium, increasing proportionally with embryo development stage. Our results suggest blastocyst synthesis of the growth factor rather than residual production by the very few corona cells that are still attached to the embryo until 6–8 cells development; furthermore, they are in accordance with previous data [17] demonstrating VEGF mRNA on human blastocysts. The sequential use of three culture media (‘fertilization medium’ during the first 24 h, ‘cleavage medium’ on the 2nd and 3rd day, ‘blastocyst medium’ on the 4th and the 5th day) in our study guaranteed the best culture conditions to obtain the greatest number of blastocysts, in accordance with other authors [18]. In humans, embryo apposition and attachment to the maternal endometrial epithelium begins around the 7th day after fertilization, when the embryo is at blastocyst stage. The capacity of the blastocyst to synthesize VEGF could indicate a possible role for this growth factor in embryonic implantation and development. VEGF is indeed a potent angiogenetic mediator which induces neovascularization and increases vascular permeability. VEGF and its receptor have been identified in several reproductive tissues, including corpus luteum, ovarian follicles, endometrial vessels and embryonic implantation sites in mice [19]. Moreover, studies in mice confirm the great importance of this molecule in embryo development: animals with functional inactivation of one VEGF allele showed several malformations in the vascular system, resulting in death on days 11 and 12 of pregnancy. A lot of evidence indicates a role for VEGF in implantation mechanisms. In the golden hamster (Mesocricetus auratus) VEGF synthesis by decidua begins on day 5 of pregnancy, while VEGF production by the embryo begins at least on day 6 [20,21]. Our results, according to the literature, suggest a biochemical dialogue between the embryo and the endometrium. We hypothesize that endometrial receptivity can be determined by its capacity to detect the small quantity of growth factors synthesized by the embryo. The downregulation of sflt (soluble VEGF receptor which acts as a soluble antagonist) during the luteal phase may play a role in sensitizing the maternal endothelial receptors to angiogenetic stimuli secreted by the implanting embryo [22]. Furthermore, the embryo is capable
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VEGF levels in human embryo culture medium of producing cytokines such as interleukin-1 and interleukin-6 [23] which can contribute to the molecular dialogue. It can be also hypothesized that the VEGF secreted by the blastocyst may serve as a marker, helpful to identify blastocysts with high implantation potential or hypoxic blastocysts with poor implantation potential. In conclusion, for the first time, our study demonstrated active VEGF synthesis by human blastocysts using a non-invasive method which evaluated only human embryo culture medium, without risks for the embryos. References 1. Cross JC, Werb Z, Fisher SJ. Implantation and the placenta: key pieces of the development puzzle. Science 1994;266:1508– 1518. 2. Tazuke SI, Giudice LC. Growth factors and cytokines in the endometrium, embryonic development, and maternal: embryonic intersections. Semin Reprod Endocrinol 1996;14: 231–246. 3. Hoozemans DA, Schats R, Lambalk CB, Homburg R, Hompes PG. Human embryo implantation: current knowledge and clinical implications in assisted reproductive technology. Reprod Biomed Online 2004;9:692–715. 4. Nardo LG. Vascular endothelial growth factor expression in the endometrium during the menstrual cycle, implantation window and early pregnancy. Curr Opin Obstet Gynecol 2005;17:419–423. 5. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 1993;13:18–32. 6. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306–1309. 7. Agrawal R, Tan SL, Wild S, Sladkevicius P, Engmann L, Payne N, Bekir J, Campbell S, Conway G, Jacobs H. Serum vascular endothelial growth factor concentrations in in vitro fertilization cycles predict the risk of ovarian hyperstimulation syndrome. Fertil Steril 1999;71:287–293. 8. Artini PG, Fasciani A, Monti M, Luisi S, D’Ambrogio G, Genazzani AR. Changes in vascular endothelial growth factor levels and the risk of ovarian hyperstimulation syndrome in women enrolled in an in vitro fertilization program. Fertil Steril 1998;70:560–564. 9. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25. 10. Van Blerkom J, Antczak M, Schrader R. The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod 1997;12:1047–1055.
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