Asimina Tavaniotou 1, Carola Albano 1, Johan Smitz 1 and Paul Devroey 1,2

Human Reproduction Vol.16, No.4 pp. 663 667, 2001 Comparison of LH concentrations in the early and midluteal phase in IVF cycles after treatment with HMG alone or in association with the GnRH antagonist Cetrorelix Asimina Tavaniotou 1, Carola Albano 1, Johan Smitz 1 and Paul Devroey 1,2 1 Centre for Reproductive Medicine, Dutch-Speaking Free University of Brussels, Brussels, Belgium 2 To whom correspondence should be addressed at: AZ-VUB, Centre for Reproductive Medicine, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: paul.devroey@az.vub.ac.be or mtavaniotou@hotmail.com Luteinizing hormone (LH) is mandatory for the maintenance of the corpus luteum. Ovarian stimulation for IVF has been associated with a defective luteal phase. The luteal phases of two groups of patients with normal menstrual cycles and no endocrinological cause of infertility were retrospectively analysed in IVF cycles. Thirty-one infertile patients stimulated with human menopausal gonadotrophins (HMG) for IVF to whom the gonadotrophin-releasing hormone (GnRH) antagonist Cetrorelix 0.25 mg was also administered to prevent the LH surge (group I) were compared with 31 infertile patients stimulated with HMG alone (group II). Despite differences in the stimulation outcome, luteal LH serum concentrations were similar in the two groups. LH values dropped from 2.3 1 IU/l on the day of human chorionic gonadotrophin (HCG) administration to 1.1 0.7 IU/l on day HCG 2 in group I (P < 0.0001) and from 5.1 3to1.2 1.7 IU/l (P < 0.0001) in group II. In the mid-luteal phase, LH concentrations were low in both groups. Our results suggest that suppressed LH concentrations in the early and mid-luteal phase may not be attributed solely to the GnRH-antagonist administration. Pituitary LH secretion may be inhibited by supraphysiological steroid serum concentrations via long-loop feedback and/or by the central action of the exogenously administered HCG via a short-loop mechanism. Key words: GnRH antagonist/ivf/lh/luteal phase/ovarian stimulation Introduction As LH is mandatory for the maintenance and normal The administration of gonadotrophin-releasing hormone steroidogenic activity of the human corpus luteum (Casper antagonist (GnRH) Cetrorelix has been shown to be effective and Yen, 1979; Schriock et al., 1985; Mais et al., 1986), in blocking the luteinizing hormone (LH) surge in ovarian abnormal LH secretion may account for a defective luteal stimulation cycles for IVF (Diedrich et al., 1994; Albano et al., phase. The aim of this study is to investigate further the 1996; Felberbaum et al., 1996; Albano et al., 1997). possible causes of the observed decrease in serum LH In contradiction to GnRH-agonist cycles, where pituitary concentrations after GnRH-antagonist treatment, by comparfunction remains impaired for the entire length of the luteal ing patients stimulated with the association of HMG and the phase after the arrest of the agonist (Smitz et al., 1992), antagonist Cetrorelix 0.25 mg with patients stimulated with adenohypophysis maintains its responsiveness to endogenous HMG only, for IVF. GnRH stimulus after antagonist treatment (Felberbaum et al., 1995) and it was suggested that antagonist cycles may not be in need of luteal phase support (Albano et al., 1997). Materials and methods Nevertheless, an impaired luteal phase in terms of duration Two groups of patients were compared in this retrospective analysis. and/or serum progesterone concentrations was observed in In both groups, the detection of infertility caused by endocrinopathies four out of six patients stimulated with the association of or polycystic ovarian syndrome or the appearance of polycystic human menopausal gonadotrophin (HMG) and Cetrorelix ovaries in the ultrasound scan were main exclusion criteria. Patients 0.5 mg cycles with no luteal phase supplementation (Albano with premature LH rise (e.g. two consecutive measurements of LH et al., 1998). A further analysis of the luteal phases in 10 IU/l) (Devroey et al., 1995) were also excluded from the analysis. Cetrorelix 0.25 or 0.5 mg/hmg cycles showed that LH serum In the first group, 31 subjects were stimulated with a combination concentrations were reduced to almost undetectable levels two of HMG and the antagonist Cetrorelix 0.25mg. The stimulation days after the human chorionic gonadotrophin (HCG) injection protocol has been previously described in detail (Albano et al., 1996, and for the whole length of the luteal phase (Albano et al., 1999). 1997). In brief, subjects were infertile women between 23 and 37 years of age, with regular menstrual cycles (24 35 days) undergoing European Society of Human Reproduction and Embryology 663

A.Tavaniotou et al. IVF in five cycles (16%) or intracytoplasmic sperm injection (ICSI) in 26 cycles (84%). Controlled ovarian stimulation was carried out Table I. Patients and cycle characteristics after stimulation with Cetrorelix with three ampoules (225 IU) HMG (Humegon; Organon, Oss, The 0.25 mg/hmg and HMG Netherlands and Menogon; Ferring, Kiel, Germany) starting on day 2 of the menstrual cycle. The dose was adjusted individually from day Cetrorelix/HMG HMG 6 of the treatment according to oestradiol values and ultrasonographic Age 30.7 ( 4.1) 30.3 ( 2.5) follicular measurements. From day 6 of the HMG injection onwards FSH day 3 (IU/l) 7.3 ( 2.2) 7.1 ( 2.8) (day 7 of the menstrual cycle), 0.25 mg of Cetrorelix (ASTA Medica Aetiology of infertility AG, Frankfurt Main, Germany) were also administered s.c. in the Male (%) 23 (74.1) 1 (3.2) anterior abdominal wall, up to and including the last day of HMG Tubal (%) 3 (9.6) 18 (58) administration. Ovulation was induced when at least three follicles Unexplained (%) 2 (6.4) 7 (22.5) Endometriosis (%) 1 (3.2) 5 (16.1) were 17 mm in diameter, by the injection of 10.000 IU of HCG Male/endometriosis (%) 1 (3.2) (Pregnyl; Organon Oss, The Netherlands). A maximum of three Male/tubal (%) 1 (3.2) embryos was replaced 2 days after the oocyte retrieval. All the Stimulation days 10.1 ( 1.2) a 7.4 ( 0.9) subjects received luteal-phase support by means of one injection Number of ampoules 32.1 ( 6.4) b 22.5 ( 3.6) Oestradiol on the day of HCG (ng/l) 2648 ( 795) c 1476 ( 643) of 1500 IU of HCG every three days starting on the day of Number of cumulus oocyte complexes 16.8 ( 6.9) d 12.7 ( 6.97) embryo transfer. Embryos transferred 2.7 ( 0.4) 2.5 ( 0.5) The second group consisted of 31 infertile patients stimulated with Pregnancy rate/embryo transfer 9/31 11/31 HMG for IVF. The stimulation protocol has also been previously Clinical pregnancy rate/embryo transfer 8 /31 8/31 described in detail (Devroey et al., 1995). In brief, patients were 25 Biochemical 1 3 Empty sac 1 36 years of age with regular menstrual cycles. Controlled ovarian Miscarriage 1 stimulation was carried out with three ampoules (225 IU) HMG (Normegon; Organon Oss, The Netherlands and Metrodin; Serono, The values are expressed as mean ( SD). Geneva, Switzerland) starting on day 2 or 3 of the menstrual cycle. a P 0.0001. After 4 days of treatment the dose was adjusted individually according b P 0.0001. P 0.0001. to serum oestradiol concentrations and ultrasound follicular measure- d P 0.02. ments. Ovulation was induced by 10 000 IU of HCG (Pregnyl; Organon Oss, the Netherlands) when three follicles 17 mm were detected by ultrasonography. The luteal phase was supplemented with HCG 1500 IU every 3 days starting 2 days after the embryo transfer. In both groups, intensive hormonal monitoring was performed through daily blood samples during the peri-ovulatory period for the detection of a premature LH surge until the day of embryo transfer. Serum gonadotrophins were measured by specific monoclonal immunoradiometric assays (IRMA) for follicle stimulating hormone (FSH) and LH and expressed in IU/l (conversion factor to SI unit 1.00; First International Reference preparation for LH 68/40 and Second International Reference preparation for FSH 78/549). The LH assay had a sensitivity of 0.3 IU/l and within- and between-assay coefficients of variation of 7 and 9% respectively. Steroid serum concentrations were expressed in ng/l for oestradiol and µg/l for progesterone Figure 1. Oestradiol serum concentrations in Cetrorelix (conversion factor to SI unit 3.671 for oestradiol and 3.180 for 0.25 mg/hmg and HMG cycles. Oestrogen values were constantly progesterone). higher in the Cetrorelix 0.25 mg group, corresponding with the Clinical pregnancy was defined as the presence of a gestational higher number of administered ampoules and retrieved cumulus sac in the ultrasound scan at 7 weeks. Data were analysed by means oocyte complexes. Eight days after the HCG, oestrogen values were similar in both groups. *P 0.05 between the two groups. of a Wilcoxon rank sum test using MedCalc software statistical program (MedCalc Software; Mariakerke, Belgium). Values are expressed as mean SD. Statistical significance was defined as a P protocols. In the Cetrorelix 0.25 mg group, significantly higher value of 0.05. oestrogen concentrations were observed during the 4 days preceding HCG administration, on the day of HCG and 4 days after the HCG (Figure 1, Table II). Progesterone serum Results concentrations were higher in the HMG group before the Patients age and day 3 FSH levels were comparable in both ovulatory HCG, but after the HCG injection progesterone groups. In group I (HMG/Cetrorelix 0.25 group) the mean age values were higher in the Cetrorelix 0.25 mg/hmg group of the patients was 30.7 4.1 years and in group II (HMG (Figure 2, Table II). FSH serum concentrations were lower in group) it was 30.3 2.5 years. Although cycle characteristics the antagonist protocol before the HCG injection, but did not were different between the two groups, the number of transferred attain statistically significant differences on any of the days embryos was similar (2.7 0.4 in the HMG/Cetrorelix studied (data not shown). 0.25 mg and 2.5 0.5 in the HMG group), resulting in eight LH values were higher in the HMG group than in the clinical pregnancies per group (pregnancy rate/embryo transfer Cetrorelix group 2 days prior to administration of the HCG, of 25.8%) (Table I). but this reached statistically significant differences 1 day Steroid serum values were different between the two before HCG administration (5.6 2.9 versus 2.5 1.2 IU/l) 664

LH concentrations during ovarian stimulation Table II. Steroid serum concentrations during the peri-ovulatory phase of the Cetrorelix 0.25 mg/hmg and HMG groups Day Progesterone (µg/l) Oestradiol (ng/l) HMG Cetrorelix HMG Cetrorelix mean 0.25/HMG mean 0.25/HMG ( SD) mean ( SD) ( SD) mean ( SD) 2 0.4 ( 0.3) 0.2 ( 0.1) 610 ( 254) 1316 ( 403) 0 0.6 ( 0.4) 0.4 ( 0.2) 1476 ( 643) 2648 ( 795) 2 6.1 ( 3.8) 11.4 ( 5.7) 975 ( 497) 1594 ( 513) 4 35 ( 15) 50.9 ( 9.8) 477 ( 287) 719 ( 249) Day 0 the day of ovulatory HCG. Figure 2. Progesterone serum concentrations in Cetrorelix 0.25 mg/hmg and HMG cycles. Progesterone concentrations were higher in the HMG group before the ovulatory HCG injection, but higher in the Cetrorelix group in the early luteal phase. Values were Figure 4. LH serum concentrations in pregnant and non-pregnant patients after treatment with Cetrorelix 0.25 mg/hmg and HMG. No statistically significant differences in the luteal LH levels were detected between pregnant and non-pregnant patients. Pregnancy was defined as a rise in serum HCG. Day 0 day of HCG injection. values were 0.7 0.6 IU/l in the antagonist group and 0.5 similar in both groups in the mid-luteal phase. The values are 0.6 in the HMG group (Figure 3). plotted on a logarithmic scale. *P 0.05 between the two groups. No statistically significant differences were detected in LH concentrations between pregnant and non-pregnant patients for any of the days studied, before and after the HCG injection, within the Cetrorelix/HMG group or within the HMG group (Figure 4). Discussion Cycle characteristics were different between the two stimulation protocols. In the HMG/Cetrorelix 0.25 mg group, ovarian stimulation was longer, the number of administered Figure 3. LH serum concentrations in Cetrorelix 0.25 mg/hmg ampoules and the number of retrieved oocytes were signiand HMG cycles. LH values are significantly higher in the HMG ficantly higher. This may be explained by the fact that as cycles one day before and on the day of HCG. Two days after the GnRH antagonists successfully prevent a premature LH surge, HCG, LH values drop significantly in both groups, reaching almost undetectable levels. *P 0.05 between the two groups. **P ovarian stimulation may be prolonged more than in cycles 0.05 versus day of HCG injection. treated without antagonists, in order to obtain a larger number of mature oocytes. (P 0.0001) and on the day of HCG (5.1 3 versus 2.3 In the pre-ovulatory phase, LH serum concentrations were 1 IU/l) (P 0.0001). One day after the ovulatory HCG, lower in the antagonist group, as antagonist treatment significantly LH serum concentrations dropped in the HMG group and reduces LH levels (Leroy et al., 1994). One day after reached a plateau or even slightly increased in the antagonist the ovulatory HCG, LH serum concentrations dropped in the group (from 2.3 1to2.5 1.9 IU/l) (Figure 3). Two days HMG group but not in the Cetrorelix group, probably due to after the HCG injection, LH values decreased in both groups. In the arrest of the antagonist. Despite the differences in cycle the Cetrorelix group, LH serum values dropped from 2.3 1 characteristics between the two protocols, the luteal phase LH IU/l on the day of HCG to 1.1 0.7 IU/l on day HCG 2 serum concentrations were similar from day 2 following the (P 0.0001) and in the HMG group they dropped from ovulatory HCG onwards. It has been previously reported from 5.1 3to1.2 1.7 IU/l (P 0.0001). For the next 4 our group that treatment with the association of HMG and days following the HCG injection, no statistically significant Cetrorelix 0.25 or 0.5 mg for ovarian stimulation reduces LH differences were detected in LH serum concentrations serum concentrations to almost undetectable levels for the between the two groups and on days 3, 4 and 8 after HCG whole length of the luteal phase in cycles supplemented with these concentrations were at almost undetectable levels in both HCG (Albano et al., 1999). Similarly, in GnRH-agonist/HMG stimulation protocols. On the 8th day after the HCG, LH cycles, undetectable LH levels and low progesterone secretion 665

A.Tavaniotou et al. in the luteal phase have been described, accounting for a of GnRH receptor gene in GT1-7 cells (Li et al., 1996) luteal-phase defect and making luteal-phase support necessary or GnRH secretion in immortalized GnRH neurons (Mores (Smitz et al., 1988). In GnRH-agonist cycles, these low LH et al., 1996). levels may be attributed to a prolonged impairment of the In stimulated cycles this assumption was supported by pituitary gonadotrophin secretory capacity after GnRH- Demoulin et al. (1991). As mid-luteal LH serum concentrations agonist treatment (Smitz et al., 1992). On the contrary, the were significantly lower in stimulated cycles compared to pituitary remains responsive to GnRH after antagonist treat- control natural cycles, the authors postulated that this might ment (Gordon et al., 1990; Felberbaum et al., 1995) and be due a possible short negative feedback from the exogenously normal corpus luteum function is preserved after mid-follicular administered ovulatory HCG. Furthermore, in cycles stimulated antagonist administration (Mais et al., 1986). Since, in our with HMG/Cetrorelix 0.5 mg and receiving no luteal phase results, LH values were strikingly similar in the early and supplementation, LH concentrations started to increase 8 days mid-luteal phase in the two groups, it may be postulated that after the HCG (Albano et al., 1998), which coincides with the this observed decrease in LH serum concentrations may not time that exogenous HCG is cleared from the circulation be attributed solely to the GnRH antagonist administration. (Mannaerts et al., 1998). On the contrary, LH concentrations Progesterone modulates LH secretion during the luteal phase remained low in cycles supplemented with HCG (Albano by influencing the LH pulse amplitude and pituitary release et al., 1999). As, in GnRH antagonist cycles, triggering of of LH (Soules et al., 1984). A short exposure to physiological ovulation is possible with a GnRH-agonist (Olivennes et al., levels of progesterone, in the range of the early luteal phase, 1996), the effect that alternative methods to induce the final has a stimulatory effect on LH secretion by acting directly oocyte maturation (e.g. GnRH-agonist or recombinant LH) at the pituitary level (Couzinet et al., 1992; Couzinet and exert on the luteal phase, remains be investigated. Schaison, 1993). On the other hand, a longer exposure to In conclusion, ovarian stimulation with virtually all the progesterone or the combined action of oestrogen and currently used stimulation protocols results in reduced LH progesterone, results in reduced frequency of LH secretion by serum concentrations in the early and mid-luteal phase. These a possible action at the hypothalamic level (Steele and Judd, low LH serum concentrations may contribute to the luteal phase 1986, 1988; Nippoldt et al., 1989). Consequently, as ovarian defect observed after ovarian stimulation. Supraphysiological stimulation results in supraphysiological steroid serum con- steroid serum concentrations may interfere with LH secretion centrations as compared to natural cycles, it may be postulated via long-loop feedback, but, additionally, the exogenously that these high steroid serum concentrations may adversely administered HCG might amplify LH secretion arrest via a effect the LH secretion by disturbing the feedback mechanisms. second short-loop negative feedback. The fact that ovarian stimulation reduces LH concentrations in the luteal phase and results in a luteal-phase defect has also been previously proposed (Messinis and Templeton, 1987). References In cycles stimulated with FSH, lower early serum LH concen- Albano, C., Smitz, J., Camus, M. et al. 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