Reproductive Biology Associates, Atlanta, Georgia, 30342, USA
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Abstract |
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Key words: embryo culture/embryo development/platelet-activating factor/pregnancy/viability
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Introduction |
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PAF production by human embryos is related to their pregnancy potential (O'Neill et al., 1987; Roudebush et al., 2001a
,b
). Since its discovery in the early 1970s (Benveniste et al., 1972
) this novel compound has been implicated in a wide variety of reproductive functions (Harper, 1989
). The exact mechanism is uncertain, yet its importance in normal fertility is significant.
PAF plays a significant role in mammalian reproduction. Exogenous PAF will stimulate early embryo metabolism and cell division (Ryan et al., 1992; Roudebush et al., 1996
; O'Neill, 1997
). Additionally, exogenous PAF will enhance implantation rates of exposed mouse embryos (Nishi et al., 1995
).
Whereas embryo-derived PAF levels have been measured in conditioned human embryo culture media (O'Neill et al., 1987), there is limited information available on its content with regard to embryo viability (as determined by pregnancy outcomes). Therefore, the study objective was to determine if the level of PAF production by human embryos correlates with pregnancy outcome following assisted reproduction.
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Study design |
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Study population
Inclusion criteria were as follows: basal FSH level <15 mIU/ml, evidence of a normal uterine cavity, and no contraindication to pregnancy. Infertility diagnoses included ovulation dysfunction, endometriosis, tubal blockage, and male factor. Controlled ovarian stimulation was initiated with leuprolide acetate (Lupron; TAP Pharmaceuticals, North Chicago, IL, USA) administered either from the midluteal phase at 0.5 mg s.c. daily until gonadotrophin injections began, at which time the Lupron dose was lowered to 0.25 mg/day until the morning of HCG injection, or as a flare protocol, in which Lupron 1.0 mg/day was were begun on cycle day 1 and continued until day 5, when the dose was lowered to 0.25 mg/day until the morning of HCG injection. Recombinant FSH (Gonal-F; Serono Laboratories Inc., Norwell, MA, USA; or Follistim; Organon Inc., West Orange, NJ, USA) was begun after pituitary down-regulation and continued until at least two follicles reached a mean diameter of 18 mm. Oocyte retrieval was scheduled 36 h after HCG injection.
Embryo culture and transfer
All culture media and protein supplements used in the study were commercially available and purchased from Irvine Scientific, Santa Ana, CA, USA. Media P1 was supplemented with 10% v/v Synthetic Serum Substitute and equilibrated in 5.0% carbon dioxide at 37°C before use. Sperm were prepared with a 90/45 discontinuous gradient wash, and the final pellet was resuspended in 500 µl P1 medium. Conventional insemination (study day 0) was performed by adding 100 000 sperm/ml to the oocytes, which were in groups of 35 in 75 µl droplets of P1 media under sterile mineral oil. Fertilization checks were carried out 1618 h after insemination. Embryos, in cohorts (15) with two pronuclei, were transferred to a new 50 µl drop of P1 medium for culture (study day 1). All embryo transfers (study day 3) were performed with a Wallace catheter (Marlow Technologies, Willoughby, OH, USA) in P1 medium. Methylprednisolone (16 mg/day) and doxycycline (100 mg twice daily) were administered for 5 days beginning on the day of oocyte retrieval. The luteal phase was supplemented with 50 mg progesterone in oil i.m. or 8% vaginal progesterone (Crinone; Serono Laboratories).
PAF content determination
Conditioned human embryo culture media were collected following conventional IVF and embryo transfer on day 3 (day 1 = pronuclear stage). An equal volume (50 µl) of 20% glacial acetic acid was added to the HECM to inactivate PAF-acetylhydrolase which is typically present (Wells and O'Neill, 1994). PAF content in HECM was determined by a PAF specific radioimmunoassay [125I] according to the manufacturer's instructions (Perkin Elmer Life Sciences, Inc., Boston, MA, USA). Briefly, primary antibodies were added to tubes containing the HECM samples, mixed and incubated for 15 min at room temperature. Secondary antibodies and tracer were added, mixed and incubated for 24 h+ at room temperature. Following centrifugation (2000 g; 30 min), supernatants were decanted and the tubes blotted and counted. The standard curve was calculated by regression analysis (logit value of normalized percentage bound versus log of ng PAF assayed). Content of PAF in the HECM is expressed as pmol/l/embryo (i.e. amount of PAF measured in the HECM/number of embryos cultured). PAF levels in the HECM were compared with pregnancy outcomes. Patients were further categorized into three groups based upon PAF levels: low (
50 pmol/l/embryo); medium (51100 pmol/l/embryo); and high (>100 pmol/l/embryo). Clinical pregnancy was defined as the presence of a fetal heartbeat on ultrasonography.
PAF radioimmunoassay performance was as follows: sensitivity (detection limit), 0.19 pmol/l; intra-assay coefficient of variation, 8.51% (determined by five replicates of samples measured in a single assay), 10.06 ± 0.29 pmol/l; inter-assay coefficient of variation 7.76% (determined by assaying same replicates in four separate assays), 10.73 ± 0.76 pmol/l.
Statistical analysis
Data, screened for normality, were analysed by Student's t-test and MannWhitney rank sum test. Statistical calculations were performed with SigmaStat for Windows, version 2.03 (Jandel Scientific Corporation, San Rafael, CA, USA). Receiveroperator characteristic (ROC) curve analyses were performed using StatTools (Chang, 2000) using PAF levels in HECM to pregnancy outcomes.
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Results |
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Discussion |
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PAF is present in uterine fluid, produced by both endometrium and preimplantation embryos. PAF uterine levels in the rabbit increase from day 35 of pregnancy (Angle et al., 1988). Embryonic-PAF production in the rabbit and mouse also increases during the preimplantation phase, with maximum levels at the expanded blastocyst stage (Minhas et al., 1993
; Ripps et al., 1993
). Embryonic-PAF can act as an autocrine stimulator of embryo development (Ryan et al., 1992
). The action(s) of this embryo-derived PAF can be blocked by PAF-antibodies or PAF antagonists (Nishi et al., 1995
; Roudebush et al., 1995
). PAF production by human embryos has been correlated with pregnancy potential, i.e. the ability for the embryos to implant successfully (O'Neill et al., 1987
; Nakatsuka et al., 1992
; Roudebush et al., 2001a
). Addition of PAF to the culture media promotes 2-cell development to the blastocyst stage in the mouse by stimulation of embryonic metabolism (Ryan et al., 1992
; Nishi et al., 1995
; Roudebush et al., 1996
; O'Neill, 1997
). The effect of PAF on mouse embryo development in vitro does, however, appear to be strain specific (Radonjic-Lazovic and Roudebush, 1995
). Improved rabbit blastocyst development after IVF with PAF-treated sperm has also been reported (Roudebush et al., 1993
).
In other cell types, the action of PAF is receptor-mediated, and this may also be true in the embryo since different PAF antagonists and PAF antibodies competitively inhibit its action (Nishi et al., 1995; Roudebush et al., 1995
). Additionally, PAF antagonists inhibit implantation (Spinks and O'Neill, 1988
; Andu et al., 1990
). These data provide further evidence on the presence and requirement of embryo-derived PAF during the pre-, peri- and implantation periods. The PAF receptor has been characterized in the mouse (Ishii et al., 1996
; Roudebush et al., 1997
). Earlier results in our laboratory suggest that temporal embryonic expression of PAF and the PAF-receptor, are related (Roudebush et al., 1998
). The embryo-derived PAF production rate peaks about the time that the mouse preimplantation embryo normally enters the uterus. The highest production level for embryonic-PAF is at the expanded blastocyst stage. Preimplantation stage embryos in a variety of species (human, mouse, sheep, rabbit and pig) produce and release PAF (O'Neill, 1987; Collier et al., 1988
; Battye et al., 1991
; Mook et al., 1998
). Furthermore, mouse preimplantation embryos cultured in the presence of PAF have enhanced developmental rates (Roberts et al., 1993
) and higher implantation rates upon transfer to synchronized recipients (Ryan et al., 1987
). This may be due to embryo-derived PAF stimulation of embryonic metabolism (Ryan et al., 1989
). PAF directly influences the oxidative metabolism of glucose and lactate in the mouse preimplantation embryo (Ryan et al., 1990
). Cholinephosphotransferase and acetyltransferase (the enzymes that catalyse the final step in the biosynthetic pathways for PAF production) are present in mouse preimplantation stage embryos (Wells and O'Neill, 1994
).
Enhanced embryo development has also been reported in rabbit oocytes fertilized in vitro with PAF-treated sperm (Roudebush et al., 1993). Preliminary results in our laboratory suggest that PAF-receptor expression is not uniform throughout the preimplantation period. Eight-cell stage embryos have a lower degree of PAF-receptor expression. This, taken with the significant increase in PAF production, suggests that PAF may regulate expression of its own receptor. In most cells, PAF binds to surface receptors inducing the formation of inositol triphosphate (IP3) and diacylglycerol (DAG) resulting in an increase of intracellular calcium (Lapetina, 1982
). Exogenous PAF affects intracellular calcium levels in mouse preimplantation embryos (Roudebush et al., 1997
; Emerson et al., 2000
). Therefore, PAF appears to bind to cell surface receptors on preimplantation embryos, initiating the formation of IP3 and DAG, and increasing intracellular calcium. As a secondary messenger, calcium can regulate preimplantation embryonic development by modulating the activity of molecules that transduce intracellular signals, which in turn influence embryonic growth and development. PAF may effect preimplantation embryo development and implantation via a receptor-mediated control of intracellular calcium. Additional studies are needed to elucidate the reproductive significance of PAF activity in preimplantation embryos and PAF's role in the establishment of pregnancy.
In summary, pregnancy outcomes may be predicted by measuring PAF levels in HECM. Additional studies are required for further development of a quick, high-throughput system to adequately determine embryonic PAF production levels by individual embryos prior to this becoming clinically routine. We are currently developing an ELISA system that will be used in a clinical trial to determine the efficacy of measuring PAF production by singleton embryos so as to select the best embryo(s) for transfer. This may allow the laboratory embryologist and/or infertility physician an opportunity to determine the embryo's viability, thus improving pregnancy potential in addition to minimizing multiple gestations.
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Notes |
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* Presented, in part, at the Annual meeting of the European Society of Human Reproduction and Embryology (ESHRE 2001), Palais de Beaulieu Congress and Exhibition Centre, Lausanne, Switzerland, July 14, 2001
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References |
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Submitted on August 1, 2001; resubmitted on October 19, 2001; accepted on January 10, 2002.