Gonadotrophin-stimulated Cycles Research Articles

Contribution of recruitable follicles to circulating anti-Müllerian hormone levels following maximal gonadotrophin stimulation in patients with limited ovarian reserve

In women, the majority of anti-Müllerian hormone (AMH) measured in serum originate from small antral follicles measuring 2–10 mm. In gonadotrophin-stimulated cycles prior to assisted reproductive technology (ART), most of the recruitable follicles develop beyond 10 mm in size and thus lose their AMH secretion capacity causing declining serum AMH levels. The aim of this study was to define the residual serum AMH level after elimination of the AMH producing recruitable follicles following maximal gonadotrophin stimulation. We measured serum AMH and number of follicles according to size at several time points during a cycle of maximal gonadotrophin stimulation (fixed dose of 300 IE HP-hMG) in 107 women with low AMH (median AMH 5 pmol/L, interquartile range (IQR) 3.3–8.3). We found that AMH decreased gradually and reached a minimum level of −55.4% (95% CI −59.6; −50.7) of the baseline value four days after ovulation trigger. Our findings suggest that the residual AMH production origins from pre-antral and small antral follicles not visible by sonography and that they account for up to 40% of the circulating AMH.

Influence of peritoneal fluid from spontaneous and stimulated cycles on sperm motility in vitro.

Peritoneal fluids (PFs) from spontaneous (n = 14) and gonadotrophin-stimulated cycles (n = 20) were obtained during diagnostic laparoscopy and gamete intrafallopian transfer (GIFT) procedures, respectively. The effects of these fluids on the linear component of sperm motility and on the percentage of motile spermatozoa were studied in vitro by objective motility assessments and compared to a control medium (B2-Ménézo). Overall, the two types of PFs were found to have rather similar effects on the motility parameters studied. However, the fluids from hormonally-stimulated cycles sustained motility better (i.e., sperm velocity and percentage of motile sperm) and in a rather constant manner as a function of time (narrower range distributions of the motility measurements). Furthermore, it was observed that under identical experimental conditions motility measurements depended not only on the type of PF used but also on the sperm sample. These results suggest that assisted reproduction procedures in which PF is the medium where the gametes eventually meet and interact, such as direct peritoneal insemination (DIPI) or peritoneal oocyte and sperm transfer (POST), could have different success rates if performed in spontaneous rather than in stimulated cycles. At the same time, our results may help to explain why different pregnancy rates were reported in different studies using DIP or POST.

Aberrant expression of angiopoietins-1 and -2 and vascular endothelial growth factor-A in peri-implantation endometrium after gonadotrophin stimulation

Ovarian stimulation affects normal endometrial development. The expression of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A) and the vascular state in the peri-implantation endometrium in women with natural and gonadotrophin-stimulated cycles were compared. The expression of these angiogenesis-associated molecules in endometrial biopsies, collected on Day 7 after human chorionic gonadotrophin injection or luteinizing hormone surge in stimulated or natural cycles respectively, or at mid-luteal phase of women undergoing diagnositic laparoscopy, were analysed. Women with gonadotrophin-stimulation had lower Ang-1, but higher Ang-2, mRNA and protein expression (P < 0.05), and increased concentrations of von Willebrand factor (vWF) and blood vessel density than those with natural cycles (P < 0.05). Although stimulated cycles had higher VEGF-A mRNA expression (P = 0.023), VEGF-A protein expression was similar between the groups. Lower Ang-1/Ang-2 but higher Ang-2/VEGF-A mRNA ratios (P = 0.025) were found after gonadotrophin-stimulation. The ratios were negatively (P < 0.001) and positively correlated (P < 0.001) with estradiol levels, respectively. Cyclical changes in Ang-1 and Ang-2, but not in VEGF-A expression were noted. The decreased Ang-1 concentration and Ang-1/Ang-2 ratio and the increased Ang-2 concentration, with the increased vWF concentration and blood vessel density, in stimulated cycles suggests advanced endometrial angiogenesis after gonadotrophin-stimulation.

Optimization of the Donor Insemination Program: A Variation on the Theme

Optimization of the Donor Insemination Program: A Variation on the Theme

Quality of oocytes and embryos in patients with polycystic ovaries

Oocytes retrieved from gonadotrophin-stimulated cycles in polycystic ovarian syndrome (PCOS) patients are of different quality and different developmental potential. We hypothesized that oocyte quality and fertilization potential might be related to the follicular fluid active renin and vascular endothelial growth factor (VEGF) concentrations. In this prospective study 20 patients with PCOS undergoing in vitro fertilization (IVF) (conventional IVF or intracytoplasmic sperm injection—ICSI) were included. Each follicular aspirate was analysed for active renin and VEGF. Oocytes and embryos were checked for quality. In empty follicular aspirates active renin concentrations were higher (mean difference: 0.05 pg/ml; range: 0.01 to 0.09 pg/ml; P=0.01) and VEGF concentrations were lower (mean difference: 169 pg/ml; range: 330 to −8 pg/ml; P=0.034) than in the aspirates containing normal appearing MII oocytes. Follicular fluid VEGF concentrations were lower in immature than in normal appearing MII oocytes (mean difference: −548 pg/ml; range: −1064 to −32 pg/ml; P=0.034). VEGF concentrations were higher in aspirates containing normal appearing MII oocytes that fertilized compared to those MII oocytes that did not (2238±691 vs. 1877±902 pg/ml; P=0.045). Differentiation of normal appearing MII oocytes that fertilize from those that do not might be the consequence of better expression and action of RAS substances and VEGF.

Maternal serum levels of placental proteins after in vitro fertilisation and their implications for prenatal screening.

To determine whether the serum levels of pregnancy-associated plasma protein A (PAPP-A), pregnancy-specific beta(1)-glycoprotein (SP1), placental lactogen (hPL) and human chorionic gonadotrophin (hCG) are different in pregnancies obtained after in vitro fertilisation (IVF) and embryo transfer (ET) in comparison to spontaneous pregnancies. Assessment of the need to establish normal medians for biochemical trisomy screening in IVF pregnancies. The population comprised 96 IVF-ET pregnancies, of which 79 came from fresh gonadotrophin-stimulated cycles and 17 from embryo transfers without gonadotrophin stimulation (natural cycle IVF, frozen embryo transfers), and 156 spontaneous pregnancies. A single blood sample was obtained between 7 + 0 and 16 + 3 weeks. PAPP-A, SP1, hPL and hCG were quantified and the levels compared between gonadotrophin-stimulated IVF, steroid-only- or non-stimulated IVF, and controls with respect to gestational age using non-parametric statistical analysis. PAPP-A and hPL levels were reduced after stimulated IVF in early gestation (before 10 pregnancy weeks); SP1 followed the same trend without reaching statistical significance. hCG tended to be increased after IVF treatment including non-gonadotrophin-stimulation cycles, and also beyond 10 pregnancy weeks. Reduced PAPP-A with increased hCG yields an increased risk in screening for foetal trisomy 21. We confirm recently published observations but do not recommend the establishment of normal medians for IVF pregnancies since the extent of the deviations is varying according to the different stimulation protocols and dosages of gonadotrophins used.

Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study.

We aimed to investigate whether sperm DNA quality may predict intrauterine insemination (IUI) outcome. The study was designed in a prospective cohort fashion, at a tertiary centre for reproductive medicine. A total of 119 patients underwent 154 cycles of IUI. Parameters related to demography, cycle management and semen sample used for IUI were evaluated. Conventional semen parameters, morphology (strict criteria), sperm DNA fragmentation and stability [evaluated by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and acridine orange staining under both acid and acid + heat denaturing conditions respectively] were measured. The main outcome measure was clinical pregnancy, defined as ultrasonographic visualization of intrauterine gestational sac(s). Logistic regression analyses were done on six sets of data, including all cycles combined, cycles with washed samples, first cycle of each couple, first cycle of each couple with washed samples, cycles stimulated with gonadotrophins and finally gonadotrophin-stimulated cycles with washed samples. The number of pre-ovulatory follicles on day of hCG, the age of the woman and the percentage of sperm with acid- + heat-resistant DNA were the parameters that predicted IUI outcome in most of these data subsets. For the gonadotrophin-stimulated cycles, age of the man appeared as a predictor as opposed to that of the woman; and for the cycles within this subgroup, where the semen sample was washed, sperm DNA fragmentation and age of the man were the only two parameters to predict IUI outcome. No samples with >12% of sperm having DNA fragmentation resulted in pregnancy. The number of follicles, age of the woman/man and sperm DNA quality may predict IUI outcome.

Results of gonadotrophin stimulation with the option to convert cycles to in vitro fertilization in cases of multifollicular development.

To avoid a high cancellation rate and/or a high multiple pregnancy rate due to multifollicular development in gonadotrophin stimulated cycles, such cycles were converted in the same cycle to in vitro fertilization/embryo transfer (IVF/ET). The results from a four year period using this strategy are summarized. Seventy-three anovulatory women (seven WHO group I, 66 WHO group II) were studied during this period. In a majority of the cycles a GnRH-analogue was used for down-regulation according to a long protocol, followed by stimulation with FSH and/or hMG. Out of 154 WHO group II gonadotrophin stimulation cycles intended for ovulation induction, 25 cycles were converted to IVF. The pregnancy and delivery rates in the IVF-converted cycles were 50% and 41%, respectively, and 31% and 22% when gonadotrophin stimulation was followed by intercourse. The cancellation rate, including both ovulation induction and IVF cycles, was 15% and the multiple pregnancy rate was 30%, mainly twins. Lean women achieved better outcome than obese women. In WHO group I only 12 cycles were performed. One cycle was converted to IVF resulting in delivery and one cycle was cancelled. The pregnancy- and delivery rates were both 50% when gonadotrophin stimulation was followed by intercourse. It is concluded that the option to convert a gonadotrophin stimulation cycle to IVF in the same cycle, in cases of multifollicular development, seemed to be a good alternative. The conversion results in a low cancellation rate and a low incidence of high order multiple pregnancies. Patients should be informed of this opportunity before entering ovulation stimulation.

Reduced pregnancy rates following the transfer of human embryos frozen or thawed in culture media supplemented with normal serum albumin.

Over a 26 month period 17% of couples having treatment in our clinical programmes selected a commercially available protein (normal serum albumin, NSA) prepared from pooled human sera instead of using their own serum as a supplement for their embryo culture media. In a retrospective analysis of >2000 gonadotrophin-stimulated cycles and 1000 cycles where frozen/thawed embryos were transferred, fertilization, embryo quality and pregnancy rates following in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) or intracytoplasmic sperm injection (ICSI) were unaffected by the type of protein used to supplement the culture medium. When embryos were thawed in medium containing NSA, both pregnancy (PR) and implantation rates (IR) were significantly lower (P <0.05) than if the medium was supplemented with serum (PR 8.3% and 17.5%; IR 4.6% and 10.5%). Inclusion of NSA before freezing reduced the IR of thawed embryos. To further test this observation all cycles where embryos were cultured and frozen in medium containing NSA (173 cycles) were matched to cycles where serum was used and the outcome was compared. At the end of 1995 just over half of the embryos in both groups had been thawed. No statistical difference was noted in the pregnancy rates (NSA, 5.6% versus serum, 11.3%) but the IR per embryo was significantly lower when embryos were cultured and frozen in medium supplemented with NSA (2.2%) than when serum was used as the supplement (6.6%).

Atrial natriuretic peptide, oestradiol and progesterone in women undergoing spontaneous and gonadotrophin-stimulated ovulatory cycles

The objective of this study was to examine the relationship between the concentrations of oestradiol and progesterone on the one hand and atrial natriuretic peptide (ANP) concentrations on the other, during the follicular and luteal phases of spontaneous and gonadotrophin-stimulated ovulatory menstrual cycles. A total of 27 ovulatory women undergoing either a spontaneous (n = 9) or a gonadotrophin-stimulated (n = 18) cycle were selected for inclusion in this study. In comparison with spontaneous cycles, gonadotrophin-stimulated cycles had increased peak follicular oestradiol (mean +/- SE; 937 +/- 150 versus 195 +/- 18 pg/ml; P < 0.05) and midluteal progesterone (mean +/- SE; 44.0 +/- 7.4 versus 14.1 +/- 2.4 ng/ml; P < 0.05) concentrations. There were no differences in the circulating ANP concentrations between the follicular and luteal phases of the menstrual cycle. Despite the increased oestradiol and progesterone concentrations following gonadotrophin stimulation, no difference in ANP concentrations was seen between stimulated and spontaneous cycles. There was no correlation between circulating concentrations of oestradiol, progesterone (at physiological and supraphysiological concentrations) and ANP throughout the menstrual cycle.

Time-dependent effects of transforming growth factor α on aromatase activity in human granulosa cells

Transforming growth factor alpha (TGF alpha) is implicated as a paracrine growth factor in the regulation of human granulosa cell function. To investigate this further, we have examined the actions of TGF alpha on the basal and follicle-stimulating hormone (FSH)-stimulated aromatase activity of human granulosa cells to determine how this growth factor influences oestrogen biosynthesis in the follicle. Granulosa cells from women having in-vitro fertilization during untreated or gonadotrophin-stimulated cycles were cultured for 1-6 days in the presence or absence of FSH or TGF alpha at a range of doses. Aromatase activity, expressed as oestradiol production, was determined after culture during a 3 h test period. After 2 days, TGF alpha (1-300 ng/ml) decreased basal and FSH-stimulated aromatase activity in a dose-dependent manner (ED50 = 3 ng/ml). In contrast, after 4 days, TGF alpha enhanced both basal and FSH-stimulated aromatase activity. Repeated experiments revealed a consistent pattern of inhibition on day 2, which was more marked in the presence of FSH (reduction by 30.6 +/- 9.1%, mean +/- SEM; n = 14; P < 0.01), and stimulation on day 4 in both the absence (increased by 61.4 +/- 20.6%, mean +/- SEM; n = 6; P < 0.05) and presence of FSH (increased by 36.0 +/- 15.2%, mean +/- SEM; n = 8; P < 0.05). The results provide further evidence that TGF alpha is a paracrine factor in the control of oestrogen biosynthesis, but the actions can be either inhibitory or stimulatory depending on the duration of exposure.

Morphometric characterization of normal and abnormal human zygotes.

Human zygotes (n = 278) from 96 in-vitro fertilization gonadotrophin-stimulated cycles were photographed in their pronuclear stage (16-18 h post-insemination). Normal morphological fertilization (two pronuclei) was observed in 215 zygotes, 17 showed only one pronucleus, 40 showed three pronuclei and six showed four. Area, perimeter and maximum and minimum diameters of each zygote and pronucleus were measured using an IBAS 2000 (Kontron) image analyser. When the four groups were compared, whole zygotes did not show any morphometric difference. However, pronuclei from these groups showed that a high number of pronuclei was directly related to small pronuclei. Differences in pronuclear size and a linear increase in the nucleus/cytoplasm relationship were found which varied with the number of pronuclei.

Triggering of ovulation in human menopausal gonadotrophin-stimulated cycles: comparison between intravenously administered gonadotrophin-releasing hormone (100 and 500 g), GnRH agonist (buserelin, 500 g) and human chorionic gonadotrophin (10 000 IU)

We studied the peri-ovulatory and luteal phases in 38 human menopausal gonadotrophin (HMG)-stimulated cycles, in which ovulation was triggered with four different i.v. bolus ovulation triggers: 100 micrograms gonadotrophin-releasing hormone (GnRH; group A, n = 9), 500 micrograms GnRH agonist (GnRHa; group B, n = 10), 10,000 IU human chorionic gonadotrophin (HCG; group C, n = 10) and 500 micrograms GnRH (group D, n = 9). Endogenous luteinizing hormone (LH) surges occurred in all cycles of groups A, B and D. The rise was slowest but highest in group B (P < 0.0001) and lowest in group A. Although the t0 serum oestradiol values were similar in all groups, day +8 oestradiol and day +4 and +8 progesterone concentrations were higher in group C (P < 0.05). At day +4 and +8, serum LH concentrations were lowest (P < 0.01) but follicle stimulating hormone (FSH) concentrations were higher. Clinically, day +8 luteal scores showed a more conspicuous degree of ovarian hyperstimulation in the HCG group (P = 0.0292). Luteal insufficiency, defined as cycles with progesterone concentrations of < 8 ng/ml, occurred much more frequently in groups A, B and D than in group C (day +4: P < 0.0003; day +8: P < 0.0001), despite progesterone supplementation. Three pregnancies (one in group C and two in group D) and one moderate case of ovarian hyperstimulation syndrome (OHSS) (in a non-conceptional group D cycle) occurred. These findings show that (i) ovulation occurs and pregnancy can be achieved following an endogenous LH surge induced by GnRH and its agonists, (ii) a high frequency of luteal insufficiency occurs in such cycles even with luteal supplementation and (iii) OHSS cannot be totally prevented by this approach, although cycles with an endogenous LH surge in general result in fewer subclinical signs of ovarian hyperstimulation.

Implantation and pregnancy rates in relation to oestradiol and progesterone profiles in cycles with and without the use of gonadotrophin-releasing hormone agonist suppression

The oestradiol (E2), progesterone (P4) levels, the corresponding E2/P4 ratios and pregnancy and implantation rates were studied in 565 in-vitro fertilization (IVF) cycles stimulated with gonadotrophins alone and in 320 cycles stimulated with adjunctive treatment of a gonadortrophin-releasing hormone agonist (GnRHa, leuprolide). The overall pattern of serum E2 and P4 levels and the E2/P4 ratios were similar in both groups, though significantly higher absolute levels were demonstrated in leuprolide cycles. Similar profiles were observed in conception and nonconception cycles within each group; significantly higher levels were noted in conception cycles starting nine days after embryo transfer. Though the overall pregnancy rate per fresh transfer in leuprolide cycles was significantly higher than in non-leuprolide cycles (32 versus 22%, P less than 0.001), this was largely due to the transfer of more embryos; the implantation rate per embryo transferred did not differ significantly between groups (11.8 versus 10.5%). We conclude that the use of GnRHa in IVF cycles increases the pregnancy rate as a consequence of more embryos being transferred, since implantation rate was unaltered by GnRHa. Further, GnRHa does not alter the basic E2 and P4 progesterone pattern observed in gonadotrophin-stimulated cycles (though the absolute levels are higher); moreover, these patterns could not be used to predict pregnancy outcome.

Follicle-stimulating hormone as a prognostic indicator in clomiphene citrate/human menopausal gonadotrophin-stimulated cycles for in-vitro fertilization

Follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestradiol and prolactin levels were studied in a sequential clomiphene citrate/human menopausal gonadotrophin (CC/HMG) regimen for in-vitro fertilization. At completion of CC administration, the median FSH level in 44 cancelled cycles was elevated compared to a control group of 65 completed cycles, 29 IU/l versus 15 IU/l, P less than 0.01. Also the median FSH/LH ratio was higher in the cancelled cycles than in the control group, 1.08 versus 0.71, P less than 0.05. Conversely, the median oestradiol level was lower in the cancelled cycles than in the completed cycles, 0.27 nmol/l versus 0.59 nmol/l, P less than 0.01. No difference was seen in the median LH and prolactin levels. An FSH value above the 95% confidence limit was found in 24 of the cancelled cycles, but in only two of the completed cycles. Based on this study, an elevated FSH value following CC administration predicts a poor response to further stimulation with an accuracy of 92.3% and should result in cancellation of the cycle.

24-h echographic profile study on follicular growth prior to laparoscopic aspiration of the graafian follicle in clomiphene citrate/human chorionic gonadotrophin-stimulated cycles

Follicular growth in the last 24 h preceding laparoscopic oocyte pick-up for in vitro fertilization purposes was studied in 46 clomiphene citrate/human chorionic gonadotrophin-stimulated cycles by means of real-time sector ultrasound. A linear increase in mean follicular diameter of 2.5 mm was established. Growth rates under monofollicular and multifollicular conditions did not differ significantly. However, the absolute dimensions of the dominant follicle in the multifollicular subgroup exceeded those observed in the monofollicular subgroup ( P < 0.01) for each observation point. A fixed CC/hCG treatment scheme results in a wide range of ultimate preovulatory follicular dimensions, without any correlation between the individual follicular size and growth rate values ( r = 0.075).