Expression of vascular endothelial growth factor mRNA in human preimplantation embryos derived from tripronuclear zygotes

FERTILITY AND STERILITY威 VOL. 74, NO. 6, DECEMBER 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Expression of vascular endothelial growth factor mRNA in human preimplantation embryos derived from tripronuclear zygotes Jan-Steffen Kru¨ssel, M.D.,a,b Barry Behr, Ph.D.,a Jens Hirchenhain, Ph.D.,b Yan Wen, M.D.,a Amin A. Milki, M.D.,a Susanne Cupisti, M.D.,b Peter Bielfeld, M.D., Ph.D.,b and Mary Lake Polan, M.D., Ph.D.a Reproductive Immunology Laboratory, Stanford University Medical Center, Palo Alto, California; and HeinrichHeine-University Medical Center, Du¨sseldorf, Germany

Received March 17, 2000; revised and accepted June 26, 2000. Supported by grant number 931614 of the North Atlantic Treaty Organization (NATO) to M.L.P. Presented in part at the 15th Annual Meeting of the European Society of Human Reproduction and Embryology, Tours, France, June 27–30, 1999. Reprint requests: Jan-S. Kru¨ssel, M.D., HeinrichHeine-University Medical Center, Dept. OB/GYN, Moorenstraße 5, D-40225 Du¨sseldorf, Germany (FAX: ⫹49 (0)211 811-8147; E-mail: kruessel@uni -duesseldorf.de). a Reproductive Immunology Laboratory, Department of Gynecology and Obstetrics, Stanford University Medical Center. b Department of Obstetrics and Gynecology, HeinrichHeine-University Medical Center. 0015-0282/00/$20.00 PII S0015-0282(00)01581-8

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Objective: To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos. Design: Human preimplantation embryos not suitable for uterine transfer were examined for ␤-actin and VEGF mRNA expression. Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion. Setting: Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Du¨sseldorf, Germany. Patient(s): Couples undergoing IVF by intracytoplasmic sperm injection for various reasons. Intervention(s): Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion. Main Outcome Measure(s): Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA. Result(s): VEGF mRNA and protein could not be detected in unfertilized oocytes. However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16 – cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts). The VEGF protein level was below the sensitivity of the ELISA. Conclusion(s): Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival. (Fertil Steril威 2000;74:1220 – 6. ©2000 by American Society for Reproductive Medicine.) Key Words: Preimplantation embryo, angiogenesis, implantation, VEGF, RT/PCR, growth factors, cytokines

After the embryo invades the maternal endometrium, its development is characterized by a dramatic growth of blood vessels coincident with decidualization, development of vascular membranes, and placental formation (1, 2). These active processes involve both angiogenesis, the growth of blood vessels sprouting from preexisting endothelium (3, 4), and vasculogenesis, the in situ formation of primordial vessels from hemangioblasts (5, 6). Vascular endothelial growth factor (VEGF) is a dimeric heparin-binding glycoprotein that has been purified as a vascular permeability factor from various tumor cell lines (7, 8) and that has been

shown to increase the proliferative ability of vascular endothelial cells in vitro (9). By alternative splicing of the mRNA, at least five different isoforms can be generated: the human VEGF-A family members have been characterized to contain 121, 145, 165, 189, and 206 amino acids (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206). Interestingly, all isoforms contain the exons 1 to 5 and 8 and differ only by various combinations of either no additional exon (VEGF121), or addition of exon 6⬘ (VEGF145), exon 7 (VEGF165), exon 6 and exon 7 (VEGF189) or exon 6, exon 6⬘, and exon 7 (VEGF206) (10 –14). Biologi-

cally, VEGF121, VEGF145, and VEGF165 are secreted forms, whereas VEGF189 and VEGF206 appear to be largely insoluble (for a review, see 15). Postimplantation mouse embryos with functional inactivation of one VEGF allele (VEGF⫾) show several malformations in the vascular system that result in lethality on days 11 and 12 of pregnancy, thus suggesting a dose-dependent regulation of fetal vascular development by VEGF (16, 17). VEGF and its receptors have also been identified in several reproductive tissues, including the corpus luteum, ovarian follicles, endometrial vessels, and embryonic implantation sites in mice (18), as well as in giant trophoblast cells and early yolk sac (19); in humans, they have been identified in the endometrium (10), placenta (13), fallopian tube, and ovary (20). Recently, VEGF mRNA and protein were detected in the human endometrium throughout the menstrual cycle with maximal expression in secretory endometrium during the luteal phase, and the protein was localized in glandular epithelial cells (21). Our previous studies showed that VEGF transmembraneous receptors KDR and FLT-1 are not regulated during the menstrual cycle in human endometrium. The soluble receptor, which acts as a competitive antagonist to VEGF was, however, down-regulated in luteal phase human endometrium (22). So far, there is no information regarding the onset of embryonic VEGF expression in the human. The aim of this study, therefore, was to examine early human preimplantation embryos during various stages of development for their expression of VEGF mRNA and VEGF protein.

MATERIALS AND METHODS Patients Patients from the Stanford University Medical Center in Palo Alto, California, were asked to participate in this study by donating one of their unfertilized oocytes or pathologically fertilized embryos for research. All patients who chose to participate signed an informed consent form; the protocol and the consent form had been previously approved by the institutional review board (Human Subjects in Medical Research Committee at Stanford University). The same downregulation/stimulation protocol (GnRH-long, FSH, hCG) was performed on all patients. To keep the possibility of a contamination with paternal mRNA as low as possible, only patients undergoing intracytoplasmic sperm injection (ICSI) were included in the study.

In Vitro Culture All embryos described in this study were fertilized in vitro by ICSI. At 24 hours after oocyte retrieval, the embryos were checked for the number of pronuclei and only tripronucleic (3PN) embryos or unfertilized oocytes were included in the study. FERTILITY & STERILITY威

Patients were stimulated using standard GnRH agonist/ FSH protocols. Ovulation was triggered when at least two follicles were 17 mm in diameter. Oocytes were retrieved transvaginally under ultrasonographic guidance 35 hours after hCG administration. All procedures were performed by the same physician. Oocytes were fertilized by ICSI 3 to 4 hours after retrieval and were cultured in groups, under mineral oil in 150-mL droplets of P1 (Irvine Scientific, Santa Ana, CA) supplemented with 10% synthetic serum substitute (SSS, Irvine Scientific) as described elsewhere (23). The embryos were then transferred to blastocyst medium (Irvine Scientific), supplemented with 10% synthetic serum substitute, and were cultured for an additional 48 to 72 hours. Oocytes or embryos from various stages of preimplantation development were examined using a modification of the methods previously described (24 –29) for their expression of ␤-actin- and VEGF-mRNAs.

Primers for Reverse Transcription and Polymerase Chain Reaction Sequences of cDNA-clones for the mRNAs that should be detected in single blastomeres (␤-actin [30], VEGF [31]) were obtained from the GenBank Database of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (Internet address: http://www2 .ncbi.nlm.nih.gov/cgi-bin/genbank). One set of corresponding outer primer-sequences as well as one set of corresponding inner primer-sequences for VEGF were constructed with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN) and synthesized in the Beckman Center, Stanford University Medical Center (Palo Alto, CA). The ␤-actin primers were obtained from Clontech Laboratories (Palo Alto, CA). To ensure that the product detected resulted from amplification of cDNA rather than contaminating genomic DNA, primers were designed to cross intron/ exon boundaries (25). The primer-cDNA-sequences, GenBank accession numbers, locations of primers on the cDNAs, and the sizes of the amplified fragments are listed in Table 1. For increased sensitivity, primers for VEGF had been especially designed to not differentiate between the isoforms (Fig. 1). Primers were tested with cDNA obtained from luteal phase human endometrium, known to be a source of VEGF-mRNA. Downstream (3⬘-end) primers of the outer primer-pairs were mixed and diluted in DEPC-treated H2O to a final concentration of 5␮M for each primer. This primer-mix was used for the reverse transcription (RT) reaction instead of random primers to transcribe more specific cDNA-products. For the first polymerase chain reaction (PCR), downstream (3⬘-end) and upstream (5⬘-end) primers of the outer primerpair, and for the second PCR, downstream (3⬘-end) and upstream (5⬘-end) primers of the inner primer-pair were mixed and diluted in DEPC-treated H2O to a final concentration of 5␮M for each primer. 1221

TABLE 1 Primers used for RT/(nested) PCR. cDNA

Genbank number

␤-Actin

g28251

Size of amplified fragment

Position of primers on cDNA

838 bp

294–325 1131–1100

VEGF

M32977

297 bp 187 bp

a

156–172 452–434 219–238 405–388

Sequence of oligonucleotide 5⬘3ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG33⬘ 5⬘3CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC33⬘a 5⬘3GGG CAG AAT CAT CAC GA33⬘ 5⬘3TGG TCT GCA TTC ACA TTT G33⬘a 5⬘3ATC GAG ACC CTG GTG GAC A33⬘ 5⬘3TGT GCT GGC CTT GGT GAG33⬘

Primers were also used to reverse transcribe the specific mRNA into cDNA.

Kru¨ssel. Vascular endothelial growth factor mRNA. Fertil Steril 2000.

Reverse Transcription

For each oocyte or embryo, a 17.5 ␮L RT-mastermix was prepared (4 ␮L 25 mM MgCl2 Solution, 2 ␮L 10⫻ PCRBuffer, 2 ␮L DEPC-treated H2O [dist.], 2 ␮L dATP, 2 ␮L dCTP, 2 ␮L dGTP, 2 ␮L dTTP [all Perkin-Elmer, Foster City, CA], 1.5 ␮L outer 3⬘ primer-mix) in a 0.5 mL thin-wall PCR tube (Applied Scientific, South San Francisco, CA) and covered with 50 ␮L of light white mineral oil (Sigma, St. Louis, MO).

The single oocyte or embryo was added to the RT mix allowing a carryover of culture medium of 1 ␮L. Samples were immediately heated up to 99°C for 1 minute in a DNA Thermal Cycler 480 (Perkin-Elmer) to release the total RNA and denature the proteins. Samples were cooled down to 4°C, and 0.5 ␮L RNase-Inhibitor (Perkin-Elmer) was added followed by 1.0 ␮L MuLV Reverse Transcriptase (Gibco BRL, Grand Island, NY). The RT-reaction was carried out in

FIGURE 1 Schematic illustration of the primer location on the VEGF mRNA. Primers were specifically designed to detect all VEGF isoforms for increased sensitivity. Primers crossed intron/exon-boundaries to enable detection of amplified genomic DNA. The left side shows a 2% agarose gel stained with ethidium-bromide with RT/PCR products obtained from luteal endometrium. PCR products were extracted from the gel and the sequences were confirmed.

Kru¨ssel. Vascular endothelial growth factor mRNA. Fertil Steril 2000.

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VEGF in human preimplantation embryos

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Enzyme Linked Immunosorbent Assay

TABLE 2 PCR specifications for amplication of the different cDNAs. mRNA

PCR round

Number of cycles

Annealing temperature (°C)

1st 1st 2nd

40 30 25

64 55 55

␤-Actin VEGF

One PCR cycle: denaturation at 94°C for 45 seconds, annealing at different temperatures for 45 seconds, extension at 72°C for 60 seconds. Kru¨ssel. Vascular endothelial growth factor mRNA. Fertil Steril 2000.

the DNA Thermal Cycler 480 (Perkin-Elmer) by using a program with the following parameters: 42°C, 60 min; 99°C, 5 min; 4°C, ⬁. After the reaction was complete, samples were stored at ⫺20°C until the first PCR.

Nested PCR The RT products were split for the first round of PCR. First, 2 ␮L of RT product was added to 48 ␮L of PCR-1-mix (3.4 ␮L 25 mM MgCl2 Solution, 4.7 ␮L 10⫻ PCR-Buffer, 33.25 ␮L DEPC-treated H2O [dist.], 1 ␮L dATP, 1 ␮L dCTP, 1 ␮L dGTP, 1 ␮L dTTP, 0.25 ␮L Polymerase Gold [all Perkin-Elmer], and 2.4 ␮L outer 3⬘ ⫹ 5⬘ primer-mix [5 ␮M each]). After all components were mixed in a 0.5 mL thin-wall-PCR cup, the reaction-mix was covered with 50 ␮L light white mineral oil, heated in the DNA-Thermal Cycler 480 to 95°C for 9 minutes to activate the Polymerase Gold, and the first PCR was performed according to the parameters specified in Table 2. After completion of the first PCR, the reaction was terminated at 72°C for 5 minutes and cooled down to 4°C. The first-round PCR-products were stored at ⫺20°C until the second PCR. For the second PCR for VEGF, 5 ␮L of first-round PCR-products were added to 95 ␮L PCR2-reaction-mix (7.2 ␮L 25 mM MgCl2 Solution, 9.5 ␮L 10⫻ PCR-Buffer, 65.0 ␮L DEPC-treated H2O [dist.], 2.2 ␮L dATP, 2.2 ␮L dCTP, 2.2 ␮L dGTP, 2.2 ␮L dTTP, 0.5 ␮L Polymerase Gold, and 4 ␮L inner 3⬘ ⫹ 5⬘ primer-mix [5 ␮M each]) in a thin-wall PCR-tube and covered with 50 ␮L mineral oil. Activation of the Polymerase Gold was identical with the first round of PCR. After completing the second round of PCR according to the parameters specified in Table 2, samples were stored at ⫺20°C until the agarose-gel electrophoresis was carried out.

Agarose-Gel Electrophoresis Horizontal 2% agarose-gel electrophoresis was performed in an ethidium-bromide solution (Sigma). After completion of electrophoresis, the agarose-gel was analyzed on the GelDoc 1000 system (Bio-Rad Laboratories, Hercules, CA). The cDNA-size calculation and densitometry was performed with Molecular Analyst Software (Bio-Rad Laboratories). FERTILITY & STERILITY威

The blastocyst-medium (Irvine Scientific) used to culture the embryos until blastocyst stage was examined by Quantikine ELISA (R&D Systems, Minneapolis, MN). The ELISA did specifically detect human VEGF165 protein; the minimum detectable level was 5 pg/mL. Culture media (50 ␮L each) from four blastocysts at a time were pooled to get the volume necessary for the assay.

RESULTS VEGF-mRNA Expression

The mRNA for ␤-actin was detected in all six oocytes and 33 preimplantation embryos, suggesting embryonic viability until cell lysis, immediately prior to the RT reaction. We could not detect VEGF mRNA in any of the six oocytes examined, meaning that no maternally synthesized mRNA is carried over into the oocyte. In 30 of the 33 embryos, however, VEGF mRNA was expressed at detectable levels. Figure 2 shows a 2% agarose gel stained with ethidiumbromide from the PCR products obtained from a hatched blastocyst. Within the groups, VEGF mRNA was detected in 5/5 hatched blastocysts, 11/12 early blastocysts, 3/4 morulae, and 11/12 8-to-16 – cell embryos (Fig. 3). This suggests that the VEGF mRNA detected in this experiment does truly reflect an embryonic synthesis and not a carryover from previously transcribed maternal RNA.

VEGF-Protein Expression No VEGF protein could be detected in any of the media examined from the 16 blastocysts, so it is presumed to be below the detectable level of the assay used.

DISCUSSION Successful fertilization has been reduced to a minor problem in human IVF since the advent of assisted reproductive techniques such as ICSI. Pregnancy and baby take-home rates, however, remain unsatisfactorily low. Enhanced culture conditions leading to the possibility of blastocyst transfer and/or multiple embryo transfers do increase pregnancy rates (32, 33) but also result in increased numbers of multiple gestations. It is clear, moreover, that multiple gestations are not only a main reason for premature deliveries, but they also have a high economic impact (34, 35); for these reasons, there is consensus that they should be avoided whenever possible. Therefore, attempts have been made to overcome these obstacles by reducing the number of embryos transferred (23, 36), but too little is known about preimplantation embryo development and the implantation process to allow preselection of viable embryos. The preimplantation embryo produces several factors during its development that signal its presence to the maternal organism. Appropriate interaction between the preimplantation embryo and maternal endometrium is at least partly 1223

during early embryonic development is needed to improve the outcome of human IVF.

FIGURE 2 The 2% agarose gel stained with ethidium-bromide with RT/(nested) PCR products from a human hatched blastocyst.

VEGF has been shown to play a crucial role during the postimplantation period of embryo development. The ligand and its receptors have been demonstrated in embryonic implantation sites in mice (18) and in the human placenta (13). More importantly, functional inactivation of one VEGF allele in mice (VEGF⫾) leads to lethality on days 11 and 12 of pregnancy as a result of vascular malformations (16, 17). The present study is the first to demonstrate the activation of the VEGF gene as early as the eight-cell stage during preimplantation embryo development in the human. Because the embryonic genome is known to become active around the four-cell stage (43, 44), VEGF appears to be among the first actively transcribed genes. In the context of our previous data on the down-regulation of the soluble VEGF-receptor, sflt, in human endometrium during the luteal phase (22), our findings in this study suggest a possible interaction between small amounts of embryonically synthesized VEGF and the endometrial KDR and Flt-1 receptors.

Kru¨ssel. Vascular endothelial growth factor mRNA. Fertil Steril 2000.

controlled by paracrine cytokines and growth factors (for reviews, see refs 27, 37– 40). Cytokine mRNAs and growth factor mRNAs have been detected in blastomeres and in preimplantation embryos from different species (24 –26, 28, 41, 42), as well as in the human endometrium throughout the menstrual cycle. A better understanding of these factors

FIGURE 3 Percentage of human preimplantation embryos with detectable expression of VEGF mRNA. All of the oocytes and embryos examined expressed ␤-actin mRNA.

Kru¨ssel. Vascular endothelial growth factor mRNA. Fertil Steril 2000.

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VEGF in human preimplantation embryos

Although the amount of VEGF-protein was too small to be detected in a commercially available ELISA, we could demonstrate active embryonic transcription of the VEGF gene early during preimplantation development. Assuming that this mRNA is also transcribed into protein, small amounts could be acting in a paracrine manner to induce neoangiogenesis at the implantation site. Because sflt competes with the transmembraneous receptors for VEGFs, signal transduction by binding of VEGF to the receptors should be favored in the luteal phase when the soluble antagonist sflt is down-regulated. The present study did not discriminate between the VEGF isoforms because of the need to maximize the sensitivity, thus we could not determine whether the VEGF mRNA detected in the embryos was predominantly the intracellular (VEGF189 and VEGF206) or the mainly secreted (VEGF121, VEGF145, and VEGF165) isoforms. We are currently attempting to increase the sensitivity of our RT/PCR to answer this question. For both ethical and practical reasons, we conducted our experiments on triploid embryos as assessed by visualization of three pronuclei 16 to 18 hours after IVF-ICSI. These embryos were, of course, unsuitable for embryo transfer. A crucial question regarding the biological significance of the findings described here is whether they are influenced by the triploidy of the blastomeres. Some polyploid embryos are capable of an extensive, morphologically normal development far beyond the eight-cell stage (45, 46) and occasional triploid births have been reported (47). It is also uncertain whether a tripronucleic egg results in triploid blastomeres: about 40% of tripronucleic eggs may revert to diploidy after the first mitotic cleavage (48). It therefore seems unlikely that the pattern of transcriptional activity in these embryos varies significantly from that of regularly fertilized, diploid embryos. Vol. 74, No. 6, December 2000

In summary, we believe that this is the first report of VEGF mRNA expression during early human preimplantation embryo development. We further hypothesize that, in the context of our earlier studies, VEGF produced by the human embryo may help to induce neoangiogenesis at the implantation site and may therefore be important to embryonic survival and successful development.

21.

22.

23. 24. Acknowledgments: The authors thank Monika Branch, Janice Gebhardt, and Douglas Moore from the Stanford University IVF-Laboratory and Gertrud Fenkes and Isabel Sonnerat from the Heinrich-Heine-University Medical Center IVF-Laboratory for their contributions.

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