1 Reproductive Medicine Unit and 2 Department of Obstetrics and Gynaecology, University of Bologna and 3 Department of Obstetrics and Gynaecology, University of Modena, Italy
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Abstract |
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Key words: controlled ovarian hyperstimulation/Doppler/IVF/L-arginine/nitric oxide
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Introduction |
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The regulation and significance of ovarian and uterine haemodynamics in human reproductive pathophysiology is becoming an important research area, and transvaginal colour flow Doppler ultrasound facilitating the detection of small vessels and the measurement of impedance to flow in the uteroovarian circulation may represent an important tool for studying the female reproductive system and pelvic haemodynamics.
An increased vascularization of ovarian follicles during the course of their development occurs in experimental animals (Koning et al., 1989). In women, an enhanced vascularization seems to be responsible for the selection and maturation of follicles both in spontaneous and stimulated IVF cycles (Weiner et al., 1993
; Balakier and Stronell, 1994
; Bassil et al., 1997
). Gonadotrophins, steroids, prostaglandins and other vasoactive molecules are involved in the regulation of ovarian blood flow (Taymor, 1996
). The importance of nitric oxide (NO) as an intra- and intercellular modulator has been recognized in many biological processes, including ovarian physiology (Anteby et al., 1996
). NO is a labile and diffusible molecule which forms stable oxidized metabolites (nitrite/nitrate; NO2-/NO3-) detectable in many biological fluids. In vivo, NO is formed from L-arginine either by a constitutive calcium-dependent, or a pro-inflammatory cytokine-inducible, NO synthase (Moncada et al., 1991
). Although the precise role of NO has not been elucidated, it has been suggested that it is involved in follicular maturation and ovulation (Anteby et al., 1996
; Tao et al., 1997
). It has also been suggested that NO may participate in periovulatory vasodilatory modulation of rat ovarian blood flow (Ben-Shlomo et al., 1994
).
The role of NO in IVF has been recently evaluated (Manau et al., 2000). These authors showed a lack of relationship between intrafollicular nitrite/nitrate concentrations and ovarian response and IVF outcome (i.e. fertilization and pregnancy rate). However, in a previous paper (Battaglia et al., 1999
), it was shown that oral L-arginine supplementation during controlled ovarian hyperstimulation in poor responder patients decreases blood flow resistance in both perifollicular and uterine arteries. Hence it was speculated that L-arginine, by modulating the permeability of follicular epithelium to plasma proteins and increasing uterine perfusion, might improve ovarian response, endometrial receptivity and pregnancy rate.
The aim of the present study was to evaluate, prospectively, blindly and randomly, the possible role of orally administered L-arginine in modifying vascular parameters and improving ovarian response to gonadotrophins in IVF cycles in normally responding women.
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Materials and methods |
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The mean (± SD) age of the patients was 33.8 ± 3.1 years (range 2837), and the mean duration of infertility was 3.7 ± 2.4 years (range 26). All patients were selected from women who suffered from tubal infertility. All had regular menstrual cycles (28 ± 4 days), and their partners were fertile according to World Health Organization standards. Patients with concurrent illness were excluded from the study. Other exclusion criteria included body mass index [BMI = weight (kg)/height (m)2] 30, endometriosis, ovarian functional cyst, polycystic ovarian syndrome, unilateral ovarian resection or ovariectomy. Likewise, patients who took regular exercise, were heavy smokers (>10 cigarettes/day), and were hypertensive (systolic blood pressure >140 mmHg and/or diastolic pressure >90 mmHg) were excluded from the study. None of the women had received hormonal treatments for at least 4 months before the IVF attempt.
In order to assess ovarian reserve, peripheral blood was obtained from all patients between 08:00 and 11:00 on day 3 of the cycle preceding the IVF attempt, after an overnight fast. Basal plasma estradiol (E2), FSH and LH concentrations were determined using a radioimmunoassay (RIA; Radim, Pomezia, Italy).
Patients were assigned randomly to two different stimulation protocols: a long GnRH agonist protocol and pure (p)FSH plus oral L-arginine (group I; n = 18); or a long GnRH agonist protocol and pFSH plus placebo (group II; n = 19). The placebo resembled (in terms of ampoule appearance, smell and flavour) the corresponding L-arginine preparation, and both the participating women and investigators were unaware which treatment was received. Randomization was performed by opening sequentially numbered sealed envelopes containing treatment allocation determined by a random number table.
Controlled ovarian hyperstimulation was achieved by an i.m. injection on day 20 of the cycle of GnRH agonist triptorelin (Decapeptyl 3.75; Ipsen, Milan, Italy) and, after pituitary desensitization (plasma E2 concentration <100 pmol/l; ovaries with no follicles >5 mm in diameter and endometrial thickness <5 mm), i.m. administration of pFSH (Metrodin 75 HP; Serono, Rome, Italy; 225 IU in the first 3 days of the cycle, then in an individually assessed dosage).
Patients were supplemented with 2x4 ampoules/day of either oral L-arginine (Bioarginina; Damor, Napoli, Italy; one ampoule = 2 g L-arginine) or placebo.
The IVF cycles were cancelled when E2 plasma levels were <1.1 pmol/l and/or fewer than three follicles were recruited by cycle day 8. Similarly, the IVF cycles were cancelled in those patients at risk of ovarian hyperstimulation syndrome (15 follicles per ovary and/or plasma E2 levels
9000 pmol/l).
When at least two follicle >17 mm in diameter were present, triptorelin, pFSH and L-arginine or placebo were withdrawn and 10 000 IU HCG (Profasi; Serono) were administered i.m. Ultrasonographic oocyte recovery was performed transvaginally 3536 h after HCG injection. The retrieved oocytes were classified as mature, immature or atretic on the basis of the morphology and appearance of the oocyte cumuluscorona complex according to published criteria (Acosta et al., 1984). In order to study the impact of embryo quality on implantation, the embryos were graded morphologically before replacement. The embryos were scored as follows: grade A, equal-sized blastomeres, no fragmentation; grade B, equal- or unequal-sized blastomeres, <20% fragmentation; grade C, equal- or unequal-sized blastomeres, 2050% fragmentation; and grade D, equal- or unequal-sized blastomeres, >50% fragmentation. Embryo transfer was performed 72 h after oocyte retrieval. Between one and three embryos were replaced at the 6- to 12-cell stage. Transcervical transfer was carried out using a Frydman catheter (SCS International, Genoa, Italy). The remaining cleaved embryos with <20% fragmentation were allocated to a cryopreservation protocol. Vaginal progesterone (Esolut; Angelini, Rome, Italy) was prescribed as luteal phase support until the serum ß-HCG assay was performed. A clinical pregnancy was diagnosed by ultrasonographic evidence of embryonic heart activity.
During the ovarian stimulation regimen the patients were submitted to hormonal (E2), biochemical (L-arginine) and ultrasonographic (follicular number and diameter, endometrial thickness) and Doppler (uterine and perifollicular arteries) evaluations. Plasma and follicular fluid concentrations of NO2-/NO3- were assayed.
Ultrasound and Doppler examinations
Transvaginal ultrasonographic assessments of endometrial thickness were performed on days 1 and 8 of ovarian stimulation, and on the day of HCG administration in both groups, using a 6.5 MHz vaginal transducer (A4 Idea; Esaote, Milan, Italy). Measurements of follicular size were performed daily beginning on day 8 of the cycle until the day of oocyte retrieval. A modified ovarian synchrony index (OSI = number of follicles >17 mm/number of follicles 1014mmx100) (Franco et al., 1994) was calculated.
Doppler flow measurements of uterine and perifollicular arteries were performed transvaginally with a 6.5 MHz (A4 Idea) colour Doppler system. The Doppler examination was performed at the beginning of pFSH administration, on day 8 of controlled ovarian hyperstimulation and on the day of oocyte retrieval. All patients were studied between 08:00 and 11:00 in order to exclude the effects of circadian rhythm on blood flow (Zaidi et al., 1995b). Patients were allowed to rest for at least 15 min before being scanned, and completely emptied their bladder in order to minimize any external effects on blood flow (Battaglia et al., 1994
). A 50 Hz filter was used to eliminate low-frequency signals originating from vessel wall movements. The maximum ultrasonographic energy was <80 mW/cm2. The intensity was within the safety limits suggested by the American Institute for Ultrasound in Medicine (Lizzi and Mortimer, 1989
). Colour flow images of the ascending branches of the uterine arteries were sampled lateral to the cervix in a longitudinal plane. The angle of insonation was altered to obtain the maximum colour intensity. When good colour signals were obtained, blood flow velocity waveforms were recorded by placing the sample volume across the vessel and entering the pulsed Doppler mode. The pulsatility index (PI), defined as the difference between peak systolic (S) and end-diastolic (D) flow velocity divided by the mean flow velocity (S D/mean) was calculated electronically. The PI has been shown to reflect blood flow impedance, and may be used when the end-diastolic frequency shift is absent or reversed. For each examination the mean value of three consecutive waveforms was obtained. No significant differences between the PI of the left and right uterine arteries were observed, and hence the average value of both arteries was used. The perifollicular arteries, starting from day 8 of controlled ovarian hyperstimulation, were identified around the follicles (>1.0 cm maximum diameter), in the ovarian stroma at the maximum distance from the surface of the ovary. Recorded spectra were analysed and the resistance index (RI) was obtained (RI = S D/S). Arteries demonstrating the lowest downstream impedance were selected for measurements, assuming that these were the branches supplying the developing follicles directly. When calculating results, the PIs of both uterine and perifollicular arteries were not corrected for heart rate. An indication of within-patient precision of the Doppler procedures was obtained by analysing the flow velocity waveforms recorded on three occasions either from uterine and perifollicular arteries at 1 min intervals. An analysis of variance of the results from 15 patients gave a mean coefficient of variation of 5.3% for uterine and 6.7% for perifollicular arteries, and showed no significant differences between the replicate analyses. Ultrasonographic and Doppler analyses were performed by one examiner (C.B.).
Hormonal and biochemical assays
Peripheral blood was obtained between 08:00 and 11:00 (after an overnight fast) on day 1, day 8, and on the day of HCG administration. The blood was immediately centrifuged, and the serum removed and stored at 70°C until taken for assay. Estradiol was measured by RIA as reported above; plasma L-arginine concentrations were assessed as described previously (Facchinetti et al., 1998).
NO production was assessed by monitoring (on day 1, day 8 and day of oocyte retrieval) plasma levels of stable oxidation products of NO metabolism (NO2-/NO3-). Since very little or no NO2- is normally found in the serum, no attempt was made to differentiate between NO2 and NO3 amounts; hence, results were reported as NO2- /NO3-. NO2-/NO3- were assayed using the Greiss reaction with previously described procedures (Clancy and Abramson, 1992; Facchinetti et al., 1997
).
NO2-/NO3- levels were also assayed in follicular fluid in those patients who reached oocyte retrieval. Following transvaginal needle aspiration of the accessible follicles, in order to homogenize the fluids and to reduce possible interfollicular differences, the follicular fluids of follicles 17 mm were pooled and immediately centrifuged (2000 g for 20 min). The supernatant was removed and stored at 70°C until bioassayed. Similarly, aliquots of follicular fluid obtained by aspiration of all accessible follicles (<17 mm) were pooled, centrifuged, stored at 70°C, and subsequently assayed. The analyses were performed using the same methods as for serum assays.
All samples from each subject were analysed in duplicate in the same assay. On the basis of two quality control samples, the average intra- and inter-assay coefficients of variation were 5.1 and 7.7% for LH, 4.8 and 7.1% for FSH, 4.9 and 7.5% for E2, and 6.8 and 11.3% for L-arginine respectively. In addition the NO2-/NO3- intra- and inter-assay coefficients of variation were 6.6 and 8.9% respectively. No differences were observed between follicular fluid and serum assays.
Statistical analysis
A statistical analysis was performed using the MannWhitney, 2, FisherIrwin exact and Wilcoxon tests and a one-way analysis of variance, where indicated. The relationship between the parameters analysed was assessed using the linear regression method. A P-value
0.05 was considered to be statistically significant. Data were presented as mean ± SD, unless otherwise indicated.
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Results |
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Discussion |
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In the present study, although the fertilization rate and the number of transferred embryos were similar in both groups, the quality (type A+B) of embryos and pregnancy rate were higher in placebo-treated patients. Furthermore, in L-arginine-treated women, the intrafollicular NO2-/NO3- concentrations were higher than in the placebo-supplemented group, whilst in the entire study population the follicular fluid NO2-/NO3- concentrations were inversely correlated with embryo quality.
Each embryo has its own developmental potential, and few cleaved embryos are competent to implant after IVF and develop through gestation (Van Blerkom et al., 1997). It is known that mature oocytes often contain chromosomal and cytoplasmic structural defects that prevent the fertilized oocytes from adequate developmental growth. How and when such anomalies intervene are not well understood. Although differences in follicle cell function and follicular fluid biochemistry have been suggested to influence the developmental potential of the human oocyte, no single factorwhether secreted into the circulation or present in the follicular fluidhas been shown to provide definitive prediction of the developmental competence of the oocyteembryo complex. It has been suggested (Gaulden, 1992
) that intrafollicular hypoxia might negatively influence spindle organization and chromosomal segregation in the human oocyte. However, the intra-ovarian regulatory system is composed of various substances including growth factors, cytokines, neuropeptides and vasoregulatory molecules and, among these, NO and its derivatives may serve an important role.
Several studies have demonstrated that elevated NO concentrations can reduce cellular ATP levels by inhibiting the cells' ATP-generating ability (Moncada et al., 1991). This cytostatic and cytotoxic mechanism induces a direct inhibition of mitochondrial respiration and DNA synthesis. Furthermore, elevated NO concentrations react actively with oxygen, yielding strongly oxidizing molecules (nitrogen dioxide and peroxy nitrites) that are potentially more toxic than NO itself (Anggard, 1994
).
The above considerations allow us to speculate that L-arginine supplementation with the consequent elevated intrafollicular NO2-/NO3- concentrations may have detrimental effects on embryo quality and pregnancy rate. This may be appear to be in contrast to a previous study, where it was affirmed that in `poor responder' patients the adjuvant L-arginine supplementation during controlled ovarian hyperstimulation improved follicular growth, oocyte quality, fertilization rate, and arguably also pregnancy rate (Battaglia et al., 1999). However, when comparing the intrafollicular content in normal and `poor' responders, it was noted that L-arginine-supplemented `poor responders' showed follicular fluid NO2-/NO3- concentrations (6.7 ± 1.11 µmol/l) (Battaglia et al., 1999
) that were similar to those in placebo-treated patients (8.68 ± 1.82 µmol/l; P = 0.064) but significantly lower than in L-arginine-supplemented, normally responding women (9.87 ± 1.35 µmol/l; P = 0.022). It was speculated that follicular fluid NO derivatives are most likely necessary for oocyte activation at fertilization and have beneficial effects when produced within physiological limits, but at higher doses they can cause cytostatic and cytotoxic effects and have detrimental consequences on embryo quality, implantation and pregnancy rate.
The pregnancy rate was significantly higher in the placebo-treated than the L-arginine-treated group, and this might be due to better embryo quality and/or improved endometrial receptivity. There are no accepted standard criteria for evaluating endometrial receptivity, although attempts have been made to correlate it with ultrasound parameters (Gonen and Casper, 1990; Khalifa et al., 1992
; Coulam et al., 1994
; Yaron et al., 1994
; Noyes et al., 1995
). In those patients who reached oocyte retrieval, similar results were obtained in terms of endometrial texture and thickness, with or without L-arginine. These data confirm that endometrial ultrasonography is not helpful in evaluating endometrial receptivity.
The measurement of impedance to uterine blood flow in IVF cycles has provided an indirect measure of endometrial receptivity (Battaglia et al., 1990; Steer et al., 1992
; Bassil et al., 1995
; Zaidi et al., 1995a
, 1996
). In the present study, a significantly lower downstream impedance in uterine arteries resulted, on the day of oocyte retrieval, in placebo-supplemented patients. These data confirm that the decrease in peripheral impedance in the uterine vascular bed, reflected by a low PI, is a consequence of increased blood flow and tissue perfusion, which may improve uterine receptivity (Goswamy et al., 1988
; Battaglia et al., 1990
, 1997
; Steer et al., 1992
).
In both groups, an inverse correlation between plasma NO2-/NO3- concentrations and uterine artery Doppler PI was seen. Furthermore, on the day of oocyte retrieval, a significantly lower uterine artery PI was observed in the placebo-treated women. Hence, it might be suggested that, in accordance with data reported by others (Ramsay et al., 1994, 1995
) who found that human uterine blood flow can be increased by the administration of a NO donor drug, the relaxation of vascular smooth muscle of endometrial vessels may be partially mediated by NO and its derivatives. However, a sudden reduction in plasma NO2-/NO3- concentration, seen after the circulation of large quantities of NO2-/NO3- for a relatively long period, might induce an intense rebound effect on vascular tone, increase the impedance to flow in the uterine vascular bed, and reduce endometrial receptivity.
The above considerations support the hypothesis that an adequate modulation of endometrial vascularity might improve the implantation and pregnancy rate.
Although further larger randomized studies are necessary to elucidate the factors that influence intra-ovarian regulation of ovarian function, it may be concluded that oral L-arginine supplementation in normally responding patients increases follicular recruitment and reduces the duration of pFSH treatment, but might also have detrimental effects on embryo quality and pregnancy rate.
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Acknowledgements |
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Notes |
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Submitted on February 2, 2001; resubmitted on August 3, 2001
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References |
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accepted on November 11, 2001.