TY - JOUR
AU - Volpe, Annibale
AB - Abstract The objective of the present study was prospectively and randomly to evaluate the role of L-arginine in improving uterine and follicular Doppler flow and in improving ovarian response to gonadotrophin in poor responder women. A total of 34 patients undergoing assisted reproduction was divided in two groups according to different ovarian stimulation protocols: (i) flare-up gonadotrophin-releasing hormone analogue (GnRHa) plus elevated pure follicle stimulating hormone (pFSH) (n = 17); and (ii) flare-up GnRHa plus elevated pFSH plus oral L-arginine (n = 17). During the ovarian stimulation regimen, the patients were submitted to hormonal (oestradiol and growth hormone), ultrasonographic (follicular number and diameter, endometrial thickness) and Doppler (uterine and perifollicular arteries) evaluations. Furthermore, the plasma and follicular fluid concentrations of arginine, citrulline, nitrite/nitrate (NO2–/NO3–), and insulin-like growth factor-1 (IGF-1) were assayed. All 34 patients completed the study. In the L-arginine treated group a lower cancellation rate, an increased number of oocytes collected, and embryos transferred were observed. In the same group, increased plasma and follicular fluid concentrations of arginine, citrulline, NO2–/NO3–, and IGF-1 was observed. Significant Doppler flow improvement was obtained in the L-arginine supplemented group. Three pregnancies were registered in these patients. No pregnancies were observed in the other group. It was concluded that oral L-arginine supplementation in poor responder patients may improve ovarian response, endometrial receptivity and pregnancy rate. Doppler, IVF, L-arginine, poor responders, ultrasonography Introduction Despite advances in assisted conception, poor ovarian response to gonadotropin stimulation remains one of the major challenges for in-vitro fertilization (IVF). The incidence is comprised of between 5% and 18% of the IVF cycles (Ben-Rafael et al., 1991; Jenkins et al., 1991). Although there is a lack of standard definitions, poor responders are patients failing to achieve an adequate number of mature follicles and/or an adequate serum oestradiol levels after gonadotrophin stimulation. This condition leads to cycle cancellation, or, in cases in which oocyte retrieval is possible, a very low pregnancy rate (Keay et al., 1997). Furthermore, a poor response identifies patients who on subsequent IVF attempts present a high cancellation rate (24–68%) (Tanbo et al., 1990). The above condition may be considered the result of diminished ovarian reserve and can be due to advanced age, prior ovarian surgery, environmental and genetic factors (Toner et al., 1991; Keay et al., 1997). Furthermore, severe endometriosis (Wardle et al., 1985) and pelvic infections (Keay et al., 1998) may impair ovarian function either through direct damage to the ovaries or through indirect mechanisms. In most patients, the inadequate responses remain unexplained (Ben-Rafael et al., 1986; Rodgers et al., 1995). For appropriate counselling and management it would be helpful to identify these patients before initiating hormonal stimulation for IVF. Advanced female age (≥40 years) (Piette et al., 1990), elevated serum oestradiol (Smotrich et al., 1995), inhibin (Halvorson and De Cherney, 1996), follicle stimulating hormone (FSH) (Muasher et al., 1988) and increased FSH/luteinizing hormone (LH) ratio (Droesch et al., 1989) on cycle day 3 have been associated with suboptimal response to stimulation. On the contrary, a rise in endogenous growth hormone (GH) secretion is associated with significantly higher plasma oestradiol concentrations and more oocytes per stimulated cycle (Stone and Marrs, 1992). Many strategies for the treatment of such patients have been proposed (Sandow et al., 1978; Check et al., 1990; Ibrahim et al., 1991; Manzi et al., 1994; Schoolcraft et al., 1997). However, despite multiple different stimulation protocols for IVF, the ideal stimulation for poor responders still remains unknown. The regulation and significance of the ovarian and uterine haemodynamics in human reproductive pathophysiology is becoming an important tool. Increased vascularization of ovarian follicles in the course of their development occurs in experimental animals (Koning et al., 1989). In women, 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). 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). The precise role of NO has not been elucidated; however, it has been thought to be involved in follicular maturation and ovulation (Anteby et al., 1996; Tao et al., 1997). It was suggested (Ben-Shlomo, 1994) that NO may participate in periovulatory vasodilatatory modulation of rat ovarian blood flow. Transvaginal colour flow Doppler ultrasound is an important tool to study the female reproductive system and pelvic haemodynamics. It facilitates the detection of small vessels in the utero–ovarian circulation and the measurement of impedance to flow in this vascular tree. The aim of the present study was prospectively to evaluate, in a group of poor responder patients, a possible role of orally administered l-arginine in modifying vascular parameters and improving ovarian response to gonadotrophins in IVF cycles. Materials and methods Patients and protocols The study protocol was approved by the local ethics review committee. In all, 34 women attending the infertility clinic participated in the study after giving informed consent. The women had previously undergone a failed IVF attempt. The IVF cycles were cancelled when oestradiol plasma concentrations were <1.100 pmol/l and/or fewer than three follicles were recruited by cycle day 8. Briefly, in cancelled cycles, ovarian stimulation was achieved as follows: mid-luteal gonadotrophin-releasing hormone analogue (GnRHa) leuprorelin acetate (Enantone 3.75; Takeda, Roma, Italy) and pure FSH (pFSH; Metrodin 75 HP; Serono, Roma, Italy); leuprorelin was administered from day 20 of the menstrual cycle (3.75 mg i.m.) and pFSH (in an individually assessed i.m. dosage) commenced after pituitary desensitization (plasma oestradiol concentration <35 mg/ml; ovaries with no follicles >5 mm in diameter and endometrial thickness <5 mm). The mean age (mean ± SD) of the above poor responder patients was 40.2 ± 2.1 years (range 37–44 years), the mean duration of infertility was 6.8 ± 3.8 (range 4–12 years). All the patients were selected among women who suffered from tubal infertility. They had regular menstrual cycles (28 ± 4 days) and their partners were fertile according to World Health Organization standards. Patients with intercurrent illness were excluded from the study. Other exclusion criteria were body mass index [BMI = weight (kg)/height2 (m2)] >30, endometriosis, ovarian functional cyst, polycystic ovarian syndrome, unilateral ovarian resection or ovariectomy. Furthermore, patients who took regular exercise, heavy smokers (>10 cigarettes/day), and with hypertension (systolic blood pressure >140 mmHg and/or diastolic pressure >90 mmHg) were excluded from the study. Women had not received hormonal treatments for at least 4 months before the first IVF attempt. To assess ovarian reserve, on day 3 of the subsequent cycle, peripheral blood was obtained from all patients between 8.00 and 11.00 a.m., after an overnight fast, and different hormonal parameters were analysed. Basal plasma oestradiol, FSH and LH concentrations were determined by a radioimmunoassay (Radim, Pomezia, Italy). The FSH/LH ratio was then calculated. Serum GH levels were similarly measured by radioimmunoassay (Sorin Biomedica, Saluggio, Italy). After a 3 month hormonal `washing-out' period, all the poor responder patients further underwent ovarian stimulation. To suppress the endogenous elevated FSH levels a combined monophasic oral contraceptive, Mercilon (20 μg ethinyl oestradiol + 150 μg desogestrel/day for 21 days; Organon Italia, Roma, Italy), was administered prior to gonadotrophin stimulation. Patients were randomly assigned to two different stimulation protocols: (i) flare-up GnRHa protocol and elevated pFSH (group I; n = 17); (ii) flare-up GnRHa protocol, elevated pFSH and oral l-arginine (group II; n = 17). Randomization was performed by opening sequentially numbered sealed envelopes containing treatment allocation determined by a random number table. Ovarian stimulation was achieved as follows: (i) group I: triptorelin (Decapeptyl 0.1; Ipsen, Milano, Italy) was daily administered from day 1 of the menstrual cycle (0.1 mg s.c.) in association with i.m. pFSH (450 IU in the first 3 days of the menstrual cycle, then in an individually assessed dosage); (ii) group II: triptorelin and pFSH were administered as in group I. In addition, patients were daily supplemented (16 g) with oral l-arginine (Bioarginina; Damor, Napoli, Italy). The IVF cycles were cancelled when oestradiol plasma levels were <1.100 pmol/l and/or fewer than three follicles were recruited by cycle day 8. When at least one follicle >17 mm in diameter was present, triptorelin, pFSH and l-arginine were withdrawn and 10 000 IU human chorionic gonadotrophin (HCG; Profasi; Serono) were administered i.m.. Ultrasonographic oocyte recovery was performed transvaginally 35 h after HCG injection. Embryo transfer was performed 48 h after oocyte retrieval and one to three embryos were replaced. Human chorionic gonadotrophin (2000 IU) was prescribed i.m. as luteal phase support on alternate days until the serum β-HCG assay. A clinical pregnancy was diagnosed by ultrasonographic evidence of embryonic heart activity. During the ovarian stimulation regimen the patients were submitted (day 0, day 8, day of HCG administration, day of oocyte retrieval) to hormonal (oestradiol, GH), ultrasonographic (follicular number and diameter, endometrial thickness) and Doppler (uterine and perifollicular arteries) evaluations. Plasma (day 0, day 8, and day of oocyte retrieval), and follicular fluid concentrations of arginine (Arg), citrulline (Cit) and nitrites/nitrate (NO2/NO3) were assayed. Furthermore, the concentration of follicular fluid insulin-like growth factor-1 (IGF-1) was also assayed. Ultrasound and Doppler examinations Transvaginal ultrasonographic assessments of endometrial thickness were performed on day 1 and 8 of ovarian stimulation, and, where possible, on the day of HCG administration in both groups, using a 6.5 MHz vaginal transducer (A4 Idea, Esaote; Milano, Italy). Measurements of follicular size were performed daily beginning on day 8 of the cycle until the day of oocyte retrieval. Doppler flow measurements of uterine and perifollicular arteries were performed transvaginally with 6.5 MHz (A4 Idea) colour Doppler system. The Doppler examination was performed at the beginning of pFSH administration, on day 8 of ovarian stimulation and on the day of oocyte retrieval. All the patients were studied between 08.00 and 11.00 h to exclude the effects of circadian rhythmicity on blood flow (Zaidi et al., 1995b). They rested for at least 15 min before being scanned, and completely emptied the bladder 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 is within the safety limits suggested by the American Institute for Ultrasound in Medicine (Lizzi and Mortimer, 1988). 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 = S–D/mean), defined as the difference between peak systolic (S) and end diastolic (D) flow velocity divided by the mean flow velocity, was calculated electronically by the machine. 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. Therefore, the average value of both arteries was used. The perifollicular arteries, starting from day 8 of ovarian stimulation, were identified around the follicles (>1.0 cm in maximum diameter), in the ovarian stroma at the maximum distance from the surface of the ovary. Recorded spectra were analysed and PI was obtained. Arteries demonstrating the lowest downstream impedance were selected for measurements, assuming that these are the branches directly supplying the developing follicles. In the results the PIs of both uterine and perifollicular arteries have not been corrected for heart rate. Ultrasonographic and Doppler analyses were performed by one examiner (C.B.) who was unaware of the patients' treatment group. Hormonal and biochemical assay Peripheral blood was obtained between 08.00 and 11.00 h, after an overnight fast, on the same day that Doppler examination took place. Blood was immediately centrifuged and serum stored at –20°C, until assays. Oestradiol and GH were measured as reported above. l-Arginine (Arg) and l-citrulline (Cit) concentrations were assessed as previously described (Facchinetti et al., 1998). Briefly, 1 ml of serum was added to 30 mg sulphosalicylic acid powder, vortexed and centrifuged at 2000 g for 20 min. A fraction of clear supernatant was mixed with an equal volume of o-pthaldialdeide for 1 min. The fluorescent derivative was then injected into a high pressure liquid chromatography apparatus equipped with a RP C-18 column. Flow rate was adjusted to 1 ml/min. The mobile phase was constituted by a mixture of 0.012 mol/l phosphate buffer with acetonitrile and methanol (91:4:5), pH 5.9. Cit and Arg had retention times of 6.8 and 10.5 min respectively. Nitric oxide production was assessed by monitoring plasma levels of stable oxidation products of NO metabolism (NO2–/NO3–). Since very little or no NO2– is normally found in the serum, we did not attempt to differentiate between the respective amounts of NO2– and NO3–; therefore, results are reported as NO2–/NO3–. The concentrations of NO2–/NO3– were assayed with the Greiss reaction with procedures previously described (Clancy and Abramson, 1992; Facchinetti et al., 1997). l-Arginine, l-citrulline, and NO2–/NO3– concentrations were also assayed in follicular fluid in those patients who reached the stage of oocyte retrieval: after transvaginal needle aspiration of the accessible follicles, the follicular fluids of follicles >17 mm were pooled and immediately centrifuged (2000 g for 20 min) and the supernatant stored at –20°C until bioassay. The analyses were performed with the same methods used for serum assays. Follicular fluid levels of IGF-1 were determined using a double-antibody radioimmunoassay (Immuno Nuclear Corp, Stillwater, USA). Statistical analysis A statistical analysis was performed using the Mann–Whitney test, χ2 test, Fisher–Irwin exact test and a one-way analysis of variance where indicated. The relationship between the parameters analysed was assessed using the linear regression method. P ≤ 0.05 was considered to be the limit of statistical significance. Data are presented as mean ± SD, unless otherwise indicated. Results All 34 patients completed the study. Hormonal evaluation, on day 3 of the cycle subsequent to the first failed IVF attempt, confirmed a poor ovarian reserve in the whole population (Table I). In the l-arginine group the cancellation rate (2/17; 11%) was lower than in group I (13/17; 76%, P < 0.001). No significant side-effects were reported by patients of l-arginine treated group. The number of pFSH ampoules, the duration of pFSH treatment, and the number of pFSH ampoules/day used in the two groups are shown in Table II, as well as the number of large follicles (>17 mm in maximum diameter), and the endometrial thickness on the day of HCG administration. The number of oocytes collected and the number of embryos transferred were significantly higher in group II than in group I (Table II). Three pregnancies (17%) were obtained in the l-arginine supplemented group, even though all of them resulted in early pregnancy loss. No pregnancies were obtained in group I patients. During ovarian stimulation, serum oestradiol and GH levels changed as indicated in Figure 1. Plasma (Figure 2) and follicular fluid (Figure 3) l-arginine, l-citrulline and NO2–/NO3– concentrations were significantly higher in group II. Follicular fluid IGF-1 concentrations were higher (Figure 3), and uterine and perifollicular blood flow resistances lower (Figure 4) in the l-arginine treated women. In the study population, plasma oestradiol concentration was highly correlated with the number of follicles (r = 0 0.819; P < 0.001), and inversely related with uterine (r = –0.503; P = 0.033) and perifollicular (r = –695; P = 0.002) artery PI. Serum GH was directly correlated with follicular fluid concentrations of l-arginine (r = 0.521; P = 0.025), and inversely correlated with uterine artery PI (r = –0.266; P = 0.044). Plasma l-arginine concentrations were correlated with the number of developed follicles (r = 0.811; P = 0.027). Plasma NO2–/NO3– concentration resulted inversely correlated with both uterine (r = –0.437; P = 0.046) and perifollicular (r = –0.487; P = 0.039) arteries PI. Follicular fluid l-arginine concentrations were inversely correlated with those in the perifollicular artery PI (r = –0.671; P = 0.024). Follicular fluid concentrations of IGF-1 were directly correlated with retrieved oocytes (r = 0.942; P = 0.017), and fertilized embryos (r = 0.849; P = 0.048). Discussion In the present study the patients, on day 3 of a spontaneous cycle, presented plasma oestradiol and FSH concentrations and FSH/LH ratio above the normal range, while plasma GH concentrations were lower than normal range. These parameters were prognostic for subsequent impaired ovarian response to ovarian stimulation, resulting, in group I, in a cancellation rate of 76%. To suppress the elevated endogenous FSH levels, both groups were treated with a cycle of oral contraceptive pill prior to gonadotrophin stimulation. In both groups, the GnRHa has been used with a flare-up protocol for taking advantage of the initial release of endogenous gonadotrophins, and, during the first 3 days of the therapy, the daily stimulation dose was increased to 450 IU pFSH. The two ovulation induction protocols, with and without l-arginine, resulted in no significant differences in the number of pFSH ampoules, the days of pFSH treatment, the number of ampoules/day and endometrial thickness. However, the numbers of large follicles (>17 mm), the number of collected oocytes and transferred embryos were significantly higher in the l-arginine group. On day 8 and on day of HCG administration, oestradiol and GH plasma concentrations were significantly increased in the treated group. Plasma l-arginine, l-citrulline and NO2–/NO3– concentrations were also significantly higher in the arginine supplemented group. In these patients oestradiol and l-arginine plasma concentrations correlated with the number of developed follicles. The above data agree with previous data (Stone and Marrs, 1992) confirming that women with increased GH concentrations produce significantly more oocytes and high oestradiol concentrations at each stage of ovarian stimulation and suggest that l-arginine supplementation may minimize poor response. Nitric oxide is an established regulator of ovarian function, and in the present study the l-arginine oral supplementation during ovarian stimulation induced an increase in plasma l-arginine, l-citrulline and nitrite/nitrate levels and was associated with increased follicular fluid concentrations of l-arginine and its derivatives. The above data were correlated with decreased blood flow resistance in the perifollicular arteries. We speculated that NO acts as vasodilator increasing the permeability of follicular epithelium to plasma proteins. The concept of the blood–follicle barrier was first introduced 40 years ago (Zachariae, 1958), when it was found that the influx of Evans blue dye was greater into stimulated pre-ovulatory follicles than into unstimulated follicles. As the follicles mature, they become more permeable to plasma proteins and result more susceptibility to circulating FSH and GH action. The simultaneous action of FSH and GH may promote an increased production of IGF-I. Growth factors such as IGF-I play a role in follicular maturation and differentiation of granulosa cells through various mechanisms (Erikson et al., 1989; Adashi et al., 1991). The intrafollicular milieu in l-arginine treated patients improved the follicular growth, and, probably, the oocyte quality and fertilization (Artini et al., 1994). Three pregnancies were observed in the l-arginine group. This result might be due to the increased number of transferred embryos, to better embryo quality, and/or to improved endometrial receptivity. No differences in embryo quality have been observed in the studied groups. There are no accepted standard criteria for evaluating endometrial receptivity, although attempts have been made to correlate ultrasound parameters and endometrial receptivity (Gonen and Casper, 1990; Dickey et al., 1992; Khalifa et al., 1992; Coulam et al., 1994; Yaron et al., 1994; Noyes et al., 1995). In those patients who reached the stage of oocyte retrieval, we obtained similar results in terms of endometrial texture and thickness with or without l-arginine. These data are in accordance with those of others, who affirmed that ultrasound determination of these parameters was not helpful in evaluating endometrial receptivity (Sterzick et al., 1991). Recently, 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 our study, a significant lower downstream impedance in uterine arteries resulted in l-arginine 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., 1989; Battaglia et al., 1990; Steer et al., 1992; Favre et al., 1993; Battaglia et al., 1997). A relationship has been found between vascularity and hormonal changes (Bassil et al., 1995). The present study showed a significant inverse correlation between uterine artery Doppler PI and plasma oestradiol concentrations. Oestradiol has been demonstrated to have effects on the vascular bed and cardiac function probably by exerting a direct effect on arterial tone (Bourne et al., 1990; de Ziegler et al., 1991; Gangar et al., 1991; Pines et al., 1991; Battaglia et al., 1995). Animal studies have demonstrated that oestrogen receptors are present in both the heart and large vessels. In addition, our data showed an inverse correlation between plasma NO2–/NO3– concentrations and uterine artery Doppler PI. Hence, we suggest that relaxation of vascular smooth muscle of endometrial vessels may be partially mediated by NO. This is in accordance with previous studies (Ramsay et al., 1994, 1995), in which it was found that human uterine blood flow, assessed using Doppler ultrasound, can be increased by administration of NO donor drug. In sheep, the administration of N-monomethylarginine (a specific inhibitor of NO) reduces by 60% the uterine artery blood flow improvements induced by oestrogens (Van Buren et al., 1992). The above considerations support the hypothesis that modulation of endometrial vascularity by oral l-arginine supplementation may improve the pregnancy rate, especially in poor responder patients who normally present an impaired uterine perfusion. Even though randomized studies are necessary to elucidate the factors that influence intra-ovarian regulation of ovarian function, the present data obtained in poor responder patients will allow better management of ovarian stimulation and may lead to more effective treatments. Table I. Ovarian reserve on day 3 of the cycle subsequent to the first failed IVF attempt   Poor responders (n = 34)  Normal range*  FSH = follicle stimulating hormone, LH = luteinizing hormone, GH = growth hormone.  *The normal range is derived from normally responsive patients attending the infertility clinic.  FSH (IU/l)  16.6 ± 8.7  1.5–10  LH (IU/l)  6.3 ± 4.2  1.5–10  FSH/LH ratio  2.6 ± 0.9  1–5  GH (μg/l)  0.8 ± 0.9  1–1.4  Oestradiol (pmol/l)  215 ± 77  <110    Poor responders (n = 34)  Normal range*  FSH = follicle stimulating hormone, LH = luteinizing hormone, GH = growth hormone.  *The normal range is derived from normally responsive patients attending the infertility clinic.  FSH (IU/l)  16.6 ± 8.7  1.5–10  LH (IU/l)  6.3 ± 4.2  1.5–10  FSH/LH ratio  2.6 ± 0.9  1–5  GH (μg/l)  0.8 ± 0.9  1–1.4  Oestradiol (pmol/l)  215 ± 77  <110  View Large Table II. Response to ovulation induction   Group I (n = 17)  Group II (n = 17)  Significance (P)  pFSH = pure follicle stimulating hormone.  No. of pFSH ampoules  35.0 ± 6.4  45.9 ± 114.6  0.543  No. of days of pFSH treatment  9.8 ± 2.2  11.4 ± 2.7  0.135  No. of ampoules/day  4.4 ± 1.2  3.7 ± 0.7  0.612  No. of follicles <17 mm  1.5 ± 0.5  5.2 ± 2.3  0.048  Endometrial thickness (mm)  0.75 ± 0.17  0.95 ± 0.21  0.144  No. of oocytes collected  1.6 ± 0.5  4.1 ± 1.9  0.049  No. of transferred embryos  1.0 ± 0.5  2.4 ± 0.5  0.50    Group I (n = 17)  Group II (n = 17)  Significance (P)  pFSH = pure follicle stimulating hormone.  No. of pFSH ampoules  35.0 ± 6.4  45.9 ± 114.6  0.543  No. of days of pFSH treatment  9.8 ± 2.2  11.4 ± 2.7  0.135  No. of ampoules/day  4.4 ± 1.2  3.7 ± 0.7  0.612  No. of follicles <17 mm  1.5 ± 0.5  5.2 ± 2.3  0.048  Endometrial thickness (mm)  0.75 ± 0.17  0.95 ± 0.21  0.144  No. of oocytes collected  1.6 ± 0.5  4.1 ± 1.9  0.049  No. of transferred embryos  1.0 ± 0.5  2.4 ± 0.5  0.50  View Large Figure 1. View largeDownload slide Serum oestradiol and growth hormone (GH) concentrations during ovarian stimulation. Group I, flare-up of gonadotrophin-releasing hormone analogue (GnRHa) protocol and elevated pure follicle stimulating hormone (pFSH) (n = 17); group II, as group I plus oral l-arginine (n = 17). Significance: *P < 0.001; aP = 0.028; bP = 0.021; cP ≤ 0.001. Figure 1. View largeDownload slide Serum oestradiol and growth hormone (GH) concentrations during ovarian stimulation. Group I, flare-up of gonadotrophin-releasing hormone analogue (GnRHa) protocol and elevated pure follicle stimulating hormone (pFSH) (n = 17); group II, as group I plus oral l-arginine (n = 17). Significance: *P < 0.001; aP = 0.028; bP = 0.021; cP ≤ 0.001. Figure 2. View largeDownload slide l-Arginine, l-citrulline, and NO2–/NO3– plasma concentrations during ovarian stimulation. HCG = human chorionic gonadotrophin. See Figure 1 for definition of groups. Significance: *P < 0.001; **P = 0.009; aP = 0.041; bP = 0.021; cP = 0.036; dP = 0.029. Figure 2. View largeDownload slide l-Arginine, l-citrulline, and NO2–/NO3– plasma concentrations during ovarian stimulation. HCG = human chorionic gonadotrophin. See Figure 1 for definition of groups. Significance: *P < 0.001; **P = 0.009; aP = 0.041; bP = 0.021; cP = 0.036; dP = 0.029. Figure 3. View largeDownload slide Follicular fluid content in patients who reached the stage of oocyte retrieval. See Figure 1 for definition of groups. Significance: aP = 0.044; bP = 0.039; cP = 0.011; dP = 0.024. Figure 3. View largeDownload slide Follicular fluid content in patients who reached the stage of oocyte retrieval. See Figure 1 for definition of groups. Significance: aP = 0.044; bP = 0.039; cP = 0.011; dP = 0.024. Figure 4. View largeDownload slide Uterine and perifollicular blood flow during ovarian stimulation. See Figure 1 for definition of groups. Significance: aP = 0.047; bP = 0.001; cP = 0.005; dP = 0.012. Figure 4. View largeDownload slide Uterine and perifollicular blood flow during ovarian stimulation. See Figure 1 for definition of groups. Significance: aP = 0.047; bP = 0.001; cP = 0.005; dP = 0.012. 3 To whom correspondence should be addressed We are grateful to Daniele Radi for expert assistance in laboratory assays. References Adashi, E.Y., Resnick, C.E., Hurwitz, A. et al. ( 1991) Insulin-like growth factors: the ovarian connection. Hum. Reprod.  , 6 , 1213–1219. Google Scholar Anteby, E.Y., Hurwitz, A., Korach, O. et al. ( 1996) Human follicular nitric oxide pathway: relationship to follicular size, oestradiol concentrations and ovarian blood flow. Hum. Reprod.  , 11 , 1947–1951. Google Scholar Artini, P.G., Battaglia, C., D'Ambrogio, G. et al. ( 1994) Relationship between human oocyte maturity, fertilization and follicular fluid growth factors. Hum. Reprod.  , 9 , 902–906. Google Scholar Balakier, H., and Stronell, R. ( 1994) Colour Doppler assessment of folliculogenesis in in vitro fertilization patients. Fertil. Steril.  , 62 , 1211–1216. Google Scholar Bassil, S., Magritte, J.P., Roth, J. et al. ( 1995) Uterine vascularity during stimulation and its correlation with implantation in in-vitro fertilization. Hum. Reprod.  , 10 , 1497–1501. Google Scholar Bassil, S., Wyns, C., Toussaint-Demylle, D. et al. ( 1997) The relationship between ovarian vascularity and the duration of stimulation in in-vitro fertilization. Hum. Reprod.  , 12 , 1240–1245. Google Scholar Battaglia, C., Larocca, E., Lanzani, A. et al. ( 1990) Doppler ultrasound studies of the uterine arteries in spontaneous and IVF stimulated ovarian cycles. Gynecol. Endocrinol.  , 4 , 245–250. Google Scholar Battaglia, C., Artini, P.G., D'Ambrogio, G. et al. ( 1994) Uterine and ovarian blood flow measurement. Does the full bladder modify the flow resistance Acta Obstet. Gynecol. Scand.  , 73 , 716–718. Google Scholar Battaglia, C., Artini, P.G., Bencini S. et al. ( 1995) Doppler analysis of uterine blood flow changes in spontaneous and medically induced menopause. Gynecol. Endocrinol.  , 9 , 143–148. Google Scholar Battaglia, C., Artini, P.G., Giulini, S. et al. ( 1997) Colour Doppler changes and thromboxane production after ovarian stimulation with gonadotrophin-releasing hormone agonist. Hum. Reprod.  , 12 , 2477–2482. Google Scholar Ben-Rafael, Z., Strauss, J.F., Mastroianni, L. and Flickinger, V.M.D. ( 1986) Differences in ovarian stimulation in human menopausal gonadotrophin treated woman may be related to follicle-stimulating hormone accumulation. Fertil. Steril.  , 46 , 586–592. Google Scholar Ben-Rafael, Z., Bider, D., Dan, U. et al. ( 1991) Combined gonadotropin releasing hormone agonist/human menopausal gonadotropin therapy (GnRHa/hMG) in normal, high, and poor responder to hMG. J. In Vitro Fert. Embryo Transf.  , 8 , 33–36. Google Scholar Ben-Shlomo, I., Adashi, E.Y. and Paync, D.W. ( 1994) The morphogenic/cytotoxic and prostaglandin stimulating activities of interleukin-1b in the rat ovary are NO independent. J. Clin. Invest.  , 49 , 1463–1469. Google Scholar Bourne, T.H., Hillard, T.C., Whitehead, M.I. et al. ( 1990) Oestrogens, arterial status and postmenopausal women. Lancet  , 335 , 1470–1471. Google Scholar Check, J.H., Nowroozi, K., Chase, J.S. et al. ( 1990) Ovulation induction and pregnancies in 100 consecutive women with hypergonadotrophic amenorrhea. Fertil. Steril.  , 53 , 811–816. Google Scholar Clancy, R.M. and Abramson, S.B. ( 1992) Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils. Anal. Biochem.  , 204 , 365–371. Google Scholar Coulam, C.B., Bustillo, M. and Soenksen, D.M. ( 1994) Ultrasonographic predictors of implantation after assisted reproduction. Fertil. Steril.  , 62 , 1004–1010. Google Scholar de Ziegler, D., Bessis, R. and Frydman, R. ( 1991) Vascular resistance of uterine arteries: physiological effects of estradiol and progesterone. Fertil. Steril.  , 55 , 775–779. Google Scholar Dickey, R.P., Olar, T.T., Curole, D.N. et al. ( 1992) Endometrial pattern and thickness associated with pregnancy outcome after assisted reproduction technologies. Hum. Reprod.  , 7 , 418–421. Google Scholar Droesch, K., Muasher, S.T., Brzyski, R.G. et al. ( 1989) Value of suppression with a GnRH-agonist prior to gonadotropin stimulation for in vitro fertilization. Fertil. Steril.  , 51 , 292–297. Google Scholar Erikson, G.F., Garzo, V.G. and Margoffin, S.A. ( 1989) Insulin-like growth factor I regulates aromatase activity in human granulosa and granulosa luteal cells. J. Clin. Endocrinol. Metab.  , 69 , 716–724. Google Scholar Facchinetti, F., De Martis, S., Neri, I. et al. ( 1997) Effects of transdermal glyceryltrinitrate on 24-h blood pressure changes in patients with gestational hypertension. Prenat. Neonat. Med.  , 2 , 22–28. Google Scholar Facchinetti, F., Valensise, H., Neri, I. et al. ( 1998) Reduction of serum citrulline levels in women at term toward the day of labor onset. Acta Obstet. Gynecol. Scand.  , 77 , 174–177. Google Scholar Favre, R., Bettahar, K., Grnage, G. et al. ( 1993) Predictive value of transvaginal uterine Doppler assessment in an in vitro fertilization program. Ultrasound Obstet. Gynecol.  , 3 , 350–353. Google Scholar Gangar, K.F., Vyas, S., Whitehead, M.I. et al. ( 1991) Pulsatility index in the internal carotid artery is influenced by transdermal oestradiol and time since menopause. Lancet  , 338 , 839–842. Google Scholar Gonen, Y. and Casper, R. ( 1990) Prediction of implantation by the sonographic appearance of the endometrium during controlled ovarian stimulation for in-vitro fertilization (IVF) J. In vitro Fertil. Embryo Transf.  , 7 , 146–152. Google Scholar Goswamy, R.K., Williams, G. and Steptoe, P.C. ( 1988) Decreased uterine perfusion a cause of infertility. Hum. Reprod.  , 3 , 955–958. Google Scholar Halvorson, L.M. and DeCherney, A.H. ( 1996) Inhibin, activin, and follistatin in reproductive medicine. Fertil. Steril.  , 66 , 459–469. Google Scholar Ibrahim, Z.H.Z., Matson, P.L., Buck, P. and Liebermann B.A. ( 1991) The use of biosynthetic human growth hormone to augment ovulation induction with buserelin acetate/human menopausal gonadotrophin in women with a poor ovarian response. Fertil. Steril.  , 55 , 202–204. Google Scholar Jenkins, J.M., Davies, D.W., Devonport, H. et al. ( 1982) Comparison of `poor' responders with `good' responders using a standard buserelin/human menopausal gonadotropin regime for in-vitro fertilization. Hum. Reprod.  , 6 , 918–921. Google Scholar Keay, S.D., Liversedge, N.H., Mathur, R.S. and Jenkins, J.M. ( 1997) Assisted conception following poor ovarian response to gonadotrophin stimulation. Br. J. Obstet. Gynecol.  , 104 , 521–527. Google Scholar Keay, S.D., Liversedge, N.H. and Jenkins, J.M. ( 1998) Could ovarian infection impair ovarian response to gonadotrophin stimulation? Br. J. Obstet. Gynaecol.  , 105 , 252–254. Google Scholar Khalifa, E., Brzyski, R.G., Oehninger, S. et al. ( 1992) Sonographic appearance of the endometrium: the predictive value for the outcome of in-vitro fertilization in stimulated cycles. Hum. Reprod.  , 7 , 677–680. Google Scholar Koning, H.E., Amselgruber, W. and Russe, I. ( 1989) La microcirculation dans les follicules et les corps jaunes d'ovaires de bovins – une atude antomique par corrosion. Contraception, Fertilité Sexualité  , 17 , 179–186. Google Scholar Lizzi, F.L. and Mortimer, A.J. ( 1998) Bioeffects considerations for the safety of diagnostic ultrasound. J. Ultrasound Med.  , 7 , 1–38. Google Scholar Manzi, D.L., Thornton, K.L., Scott, L.B. and Nulsen, J.C. ( 1994) The value of increasing the dose of human menopausal gonadotrophins in women who initially demonstrate a poor response. Fertil. Steril.  , 62 , 251–256. Google Scholar Moncada, S., Palmer, R.M.J. and Higgs, E.A. ( 1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev.  , 43 , 109–142. Google Scholar Muasher, S.J., Oehninger, S., Simonetti, S. et al. ( 1988) The value of basal and/or stimulated serum gonadotropins levels in prediction of stimulated response and in vitro fertilization outcome. Fertil. Steril.  , 50 , 98–307. Google Scholar Noyes, N., Liu, H.C., Sultan, K. et al. ( 1995) Endometrial thickness appears to be a significant factor in embryo implantation in in-vitro fertilization. Hum. Reprod.  , 10 , 919–922. Google Scholar Piette, C., deMouzon, J., Bachelot, A. and Spira, A. ( 1990) In-vitro fertilization: influence of women's age on pregnancy rates. Hum. Reprod.  , 5 , 56–59. Google Scholar Pines, A., Fiwsman, E.Z., Averbuch, M. et al. ( 1991) The effects of hormone replacement therapy in normal postmenopausal women: measurements of Doppler derived parameters of aortic flow. Am. J. Obstet. Gynecol.  , 164 , 806–812. Google Scholar Richards, J.S. and Midgley, A.R. Jr ( 1976) Protein hormone action: a key to understanding follicular and luteal cell development. Endocrinology  , 97 , 1174–1184. Google Scholar Rodgers, M., McLoughlin J.D., Lambert, A. et al. ( 1995) Variability in the immunoreactive and bioactive follicle stimulating hormone content of human urinary menopausal gonadotrophin preparations. Hum. Reprod.  , 10 , 1982–1986. Google Scholar Ramsay, B., De Belder, A., Campbell, S. et al. ( 1994) A nitric oxide donor improves uterine artery diastolic blood flow in normal early pregnancy and in women at high risk of pre-eclampsia. Eur. J. Clin. Invest.  , 24 , 76–78. Google Scholar Ramsay, B., Johnson, M.R., Leone, A.M. and Steer, P.J. ( 1995) The effect of exogeneous oestrogen on nitric oxide production in women: a placebo controlled crossover study. Br. J. Obstet. Gynecol.  , 102 , 417–419. Google Scholar Sandow, J., Von Rechenberg, W., Jerzaberg, G. and Stoll, W. ( 1978) Pituitary gonadotrophin inhibition by highly active analog of luteinizing hormone releasing hormone. Fertil. Steril.  , 30 , 205–209. Google Scholar Schoolcraft, W., Schlenker, T., Gee, M. et al. ( 1997) Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose follicle-stimulating hormone flare, growth hormone protocol. Fertil. Steril.  , 67 , 93–97. Google Scholar Smotrich, D.B., Widra, E.A., Gindoff, P.R. et al. ( 1995) Prognostic value of day 3 estradiol on in vitro fertilization outcome. Fertil. Steril.  , 64 , 1136–1140. Google Scholar Steer, C.V., Campbell, S., Tan, S.L. et al. ( 1992) The use of transvaginal color flow imaging after in vitro fertilization to identify optimum uterine conditions before embryo transfer. Fertil. Steril.  , 57 , 372–376. Google Scholar Sterzik, K., Grab, D., Rosenbush, B. et al. ( 1991) Receptivity of the endometrium: comparison of ultrasound and histological findings after hormonal stimulation. Geburtshilfe Frauenheilkd.  , 51 , 554–558. Google Scholar Stone, B.A. and Marrs, R.P. ( 1992) Ovarian responses to menopausal gonadotrophins in groups of patients with differing basal growth hormone levels. Fertil. Steril.  , 58 , 32–36. Google Scholar Tanbo, T., Abyholm, T., Bjoro, T. and Dale, P.O. ( 1990) Ovarian stimulation in previous failure from in vitro fertilization: distinction of two groups of poor responders. Hum. Reprod.  , 5 , 811–815. Google Scholar Tao, M., Kodama, H., Kagabu, S. et al. ( 1997) Possible contribution of follicular interleukin-1β to nitric oxide generation in human pre-ovulatory follicles. Hum. Reprod.  , 12 , 2220–2225. Google Scholar Taymor, M.L. ( 1996) The regulation of follicle growth: some clinical implications in reproductive endocrinology. Fertil. Steril.  , 65 , 235–247. Google Scholar Toner, J.P., Philput, C.B., Jones, G.S. and Muasher, S.J. ( 1991) Basal follicle-stimulating hormone is a better predictor of in vitro performance than age. Fertil. Steril.  , 55 , 784–791. Google Scholar Van Buren, G.A., Yang, D.S. and Clark, K.E. ( 1992) Estrogen-induced uterine vasodilatation is antagonized by l-nitroarginine methylester, an inhibitor of nitric oxide synthesis. Am. J. Obstet. Gynecol.  , 167 , 828–833. Google Scholar Wardle, P.G., McLaughlin, E.A., McDermott, A. et al. ( 1985) Endometriosis and ovulatory disorder: reduced fertilization in vitro compared with tubal and unexplained infertility. Lancet  , ii , 236–237. Google Scholar Weiner, Z., Thaler, I., Levron, J. et al. ( 1993) Assessment of ovarian and uterine blood flow by transvaginal colour Doppler in ovarian-stimulated women: correlation with the number of follicles and steroid hormone levels. Fertil. Steril.  , 59 , 743–749. Google Scholar Yaron, Y., Botchan, A., Amit, A. et al. ( 1994) Endometrial receptivity in the light of modern assisted reproductive technologies. Fertil. Steril.  , 62 , 225–232. Google Scholar Zachariae, F. ( 1958) Studies on the mechanism of ovulation: permeability of the blood-liquor barrier. Acta Endocrinol.  , 27 , 339–342. Google Scholar Zaidi, J., Campbell, S., Pitroff, R. et al. ( 1995a) Endometrial thickness, morphology, vascular penetration and velocimetry in predicting implantation in an in vitro fertilization program. Ultrasound Obstet. Gynecol.  , 6 , 191–198. Google Scholar Zaidi, J., Jurkovic, D., Campbell, S. et al. ( 1995b) Circadian variation in uterine artery blood flow indices during the follicular phase of the menstrual cycles. Ultrasound Obstet. Gynecol.  , 5 , 406–410. Google Scholar Zaidi, J., Pittrof, R. and Shaker, A. ( 1996) Assessment of uterine artery blood flow on the day of human chorionic gonadotropin administration by transvaginal color Doppler ultrasound in an in vitro fertilization program. Fertil. Steril.  , 65 , 377–381. Google Scholar © European Society of Human Reproduction and Embryology
TI - Adjuvant L-arginine treatment for in-vitro fertilization in poor responder patients
JF - Human Reproduction
DO - 10.1093/humrep/14.7.1690
DA - 1999-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/adjuvant-l-arginine-treatment-for-in-vitro-fertilization-in-poor-EG80fxS3vj
SP - 1690
EP - 1697
VL - 14
IS - 7
DP - DeepDyve
ER -