Keywords

Ovarian stimulation; IVF; Synchronic follicular growth

Introduction

Cycles of controlled ovarian stimulation (COS) with GnRH antagonists for in vitro fertilization (IVF) have become the most commonly used protocol in IVF centres. Since first studies [1] found worst IVF outcome compared to long protocols with GnRH agonists, later publications [2,3] have proved similar results in terms of clinical pregnancy rates and live birth rates. Current use of antagonist protocol is preferred because it requires less stimulation time, lower gonadotropin doses and consequently a lower cost [4]. Another important fact is that by using antagonist protocol, trigger can be done with GNRH analogues reducing significantly hyperstimulation risk [5,6].

Nevertheless, antagonist cycles tend to cause an asynchronous ovarian growth with an early dominant follicle leading and heterogeneous size follicle cohort [4]. During COS, antral follicles are required to grow co-ordinately in response to exogenous gonadotropins to accomplish simultaneous functional and morphological maturation. Marked follicular size discrepancies are related to differences in follicles sensitivity to FSH and un-satisfactory maturation. This phenomenon potentially reduces the number of viable oocytes and embryos and the probability of conception [7,8]. This asynchrony may constitute a plausible explanation for the putative poorer IVFembryo transfer (ET) outcome with GnRH antagonist detected in firsts RCT when experience with antagonists protocols was more limited [1]. Management of asynchronous cycles can be problematic since early antagonist introduction may reduce smaller follicle growth and too late antagonist initiation can cause a premature LH surge, interfere with ovocyte maturation and quality or with endocrinal milieu and endometrial receptivity. We propose a strategy in which if a leading follicle of 16 mm was detected, antagonist was delayed until E2 reached 600 pg/ml or until another follicle reached 14 mm. By ignoring the early growing follicle, we pretend to get more follicles from the cohort and to prolong the cycle and consequently to get more mature oocytes. Main objective of this study was to check whether our strategy with asynchronous cycle management was able to correct possible adverse impact of asynchrony. For that purpose, we compared those cycles to ovarian stimulation cycles were follicular growth was synchronous.

Materials and Methods

We underwent a retrospective case control study including 560 antagonist stimulation cycles in Human Reproduction Unit, Hospital 12 de Octubre in 2013. Cases were 313 antagonist cycles with a leading follicle ≥ 16 mm detected on first control, between forth or sixth stimulation days were compared with 247 controls. Controls were patients with no follicle of more than 15 mm on first control (S4-S6). First stimulation control was fixed between forth or sixth stimulation days depending of internal organisation.

Controlled ovarian stimulation cycle with GnRH antagonist protocol started on second or third day of cycle. In all patients a transvaginal ultrasound was done to check there were no follicles of more than 10 mm before starting stimulation. Gonadotropin dose was chosen according to BMI, ovarian antral follicle count, day 3 FSH; patient age or previous cycle response. We used recombinant FSH and additional LH effect (recombinant LH or HMG) was reserved for patient with suspected impaired ovarian reserve. Follicle growth was monitored every 48 or 72 h by transvaginal ultrasound; oestradiol measurement was done at each control visit.

If an asynchronous follicle was detected, antagonist was delayed until E2 reached 600 pg/ml. In control group, antagonist was introduced when main follicles got to 14 mm and E2 was above 400 pg/ml. In control group, with synchronic follicle growth, antagonist started when leading follicles were at 14 mm and E2 was above 400 pg/ml or when E2 was higher than 600 pg/ml even thaw follicles were still small.

First outcome was live birth rate and secondary ones were number of mature oocytes, fertilization rates, number of embryos; number of good quality embryos; implantation rates; cancellation rates, pregnancy rates. Variables concerning patient’s characteristics and cycle evolution were also reported.

Descriptive statistics for categorical variables were expressed as the percentage (%), whereas continuous variables were expressed as mean ± standard deviations. The Chi- square (χ2) test was used to compare categorical variables. The Student t test was used to compare continuous variables. Normality of continuous variables was verified with Shapiro-Wilk test. Variance equality was tested with levene test. A p value of less than 1.5 was considered to be statistically significant. All the statistical analyses were performed using the programme Stata 13.0 (STATA Corporation, College Station, Texas). No specific ethic approval was necessary as the study was a retrospective review design based on electronic patient’s chart review and intervention was proposed (Orden 730/2004, de 30 de junio, del Consejero de Sanidad y Consumo de la Comunidad de Madrid) . Before IVF treatment informed concerned was signed allowing medical data to be used for medical investigation.

Results and Discussion

Incidence

In our clinic, 55.89% of antagonist cycles had an early growing leading follicle measuring more than 15 mm before S6. No statistical differences were found when comparing case and control group in terms of patient’s age, day 3 FSH; antral follicle count or associated male factor. Use of hormonal contraception in previous cycle was not significantly different in the two groups (Table 1).

Table 1 Patients characteristics.

Cycle characteristics

Stimulation was longer in synchronic group (9.39 vs. 9.10 days) but no statistical significance could be detected in sense. In first control, from day 4 to day 6 stimulation day, the number of follicles over 14 mm (14-15 mm) was higher in cycles with an early dominant follicle (p<0.05). Oestradiol level in first control was logically higher in asynchronous group (p<0.05). At the end of stimulation, they were similar number of follicles of 16 mm or more, potentially having mature oocytes in both groups but cystic follicles over 22 mm were more when there had been an early dominant follicle (p<0.05). No differences were found in final E2 level (Table 2).

Table 2 Cycle characteristics.

Cycle outcome

No differences were found in oocytes number, mature oocyte rate or atresic oocytes rate. Nevertheless, a higher number of immature oocyte was observed in the control group. Fertilization rate was significantly higher in patients with an early dominant follicle (62.9% vs. 59.5%; p<0.05). Number of obtained embryos and good quality embryos, or even good quality embryo rate, were more favourable in case group but differences didn’t reach significance. An important difference was observed in implantation rate: 16.39% in control group vs. 9.47% in cases (p<0.05). Pregnancy rate, multiple pregnancy rates, miscarriage rate and live birth rate appeared to be similar in both group.

Cancelation rate was defined, as cycles where no transfer was possible: cancelled oocyte pick-up, no available embryos or cancelled embryo transfer because of hyperstimulation risk. Cancelation rate was significantly lower in asynchronous cycles: 22.98% vs. 7.96% (Table 3).

Table 3 Cycle outcome.

Discussion

Incidence

Incidence of asynchronous and fast ovarian growth was as high as 55.89% in our antagonist cycle series. Asynchrony has already been described when using antagonist stimulation protocols in which there is no endogenous down-regulation like in long agonist protocol [4]. Accelerated follicle growth has been associated to lower ovarian reserve [9] and it is important to note that in our centre 53% of our patients were classified as low ovarian reserve. Nevertheless, no difference could be found between patient’s age, antral follicular count, day 3 FSH or gonadotropin starting dose for ovarian stimulation.

A plausible mechanism for irregular follicle growth, involves the premature, gradient FSH elevation that occurs during the late luteal phase in the menstrual cycle [10]. During the lutealfollicular transition, FSH preserves early antral follicles from atresia and ensures their subsequent growth [11]. Previous oral contraception would have avoided small luteal phase FSH elevation than could determine initial growth of most sensitive follicles. Nevertheless, in our study, previous contraception treatment didn’t seem to have any effect on early follicular growth incidence.

Beside all those finding, as all patients had an ultrasound to check there was no follicle oversized (>10 mm) before COS, asynchronous follicle growth would rather correspond to an abnormal dynamic of natural folliculogenesis in our population even though we couldn’t correlate it with low ovarian reserve. Experimental studies have demonstrated a shortened follicular phase in ovulatory older women caused by a shortening in early follicular phase related to an advanced selection of dominant follicle [9].

Cycle Characteristics

Criteria for triggering are the same in all cases (more than 3 follicles of more than 18 mm), that will justify the fact that no differences were found in the number of follicles of more, 15 mm and the final E2 level and the length of stimulation. In the first control, in patients with a follicle of more 16 mm or more, it appeared more likely to find more follicles of 14-15 mm pointing at a specially accelerated folliculogenesis in that kind of patients.

Cycle outcome

Even if a slight (although not significant) shortening in cycle was observed when a big follicle was detected on first control, no differences were found in oocytes maturity or atresic rate neither in total oocyte number or metaphase II oocyte number. This could be a justified by a correct folliculogenesis in terms of oocyte maturation besides initial speed and besides the fact than a higher number of cystic follicles were detected at the end of stimulation. Maintaining the same criteria for ovulation induction should have contributed to reach similar maturity rates. Larger studies would help to confirm those findings.

The finding of higher fertilization rates in patients with an early dominant follicle (62.9% vs. 59.5%, p<0.05) may reflect a better quality oocyte but embryo quality didn’t confirm it. No significant differences were found when analysing the number of obtained embryos and good quality embryos, or good quality embryo rate. Whether a larger sample size would clarify a possible effect on oocyte and embryo quality remains to be elucidated.

Implantation impairment observed in control group could be explained by a lower endometrial receptivity as no effect on embryo quality could be observed. Previous studies found a negative correlation between progesterone levels on HCG day [12-16] and even to a chronic exposure to progesterone during follicular phase. Final follicular phase progesterone measure is related to oestradiol level [17]. A fast follicle growth could be related to higher progesterone levels since earlier in follicle phase as oestradiol appeared to be higher in first control [18,19]. This hypothesis couldn’t be confirmed, as routine progesterone was not measured in our clinical practice.

In our protocol, a very fast follicle growth was not a criterion for cancelation. This allowed us to avoid many early cancelations for mono or oligo-follicular growth, by waiting for the follicle cohort. As only 1.17 % of the cycles reaching oocyte pick-up, were cancelled because of hyperstimulation risk, we can conclude that most of the avoid cancelations are due to an insufficient ovarian response. Delaying antagonist until another follicle had reached 14 mm or E2 above 600 pg/ml, allowed us to collect oocytes with no apparent quality impairment but this strategy could have a detrimental effect on endometrial receptivity.

Conclusion

In GnRH antagonists an asynchronous follicle growth is often observed since first stimulation days with early dominant follicle over 15 mm. In such cases, we propose an expectant management for antagonist initiation in order to allow the rest of the follicle cohort to reach 14 mm or oestradiol level to be above 600 pg/ml. This strategy avoids cancelations for low response and didn’t show any impairment in oocyte and embryo quality parameters even though fertility rate was significantly better in asynchronous group. The major inconvenience was a reduction in implantation rate that could be justified by a lower endometrial receptivity. No adverse effect was observed in clinical pregnancy rates or in live birth rates. Further studies with larger series and sequential measure of progesterone levels would be needed to confirm these results. A correct management asynchronous cycle is essential to avoid cancellations and to control a possible negative effect in endometrium receptivity.

Funds

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

References

1. Al-Inany HG, Aboulghar M (2001) GNRH antagonist in assisted conception: A Cochrane review. Cochrane Database Syst Rev 4: CD001750.
2. Kolibianakis EM, Collins J, Tarlatzis BC, Devroey P, Diedrich K, et al. (2006) Among patients treated for IVF with gonadotrophins and GnRH analogues, is the probability of live birth dependent on the type of analogue used? A systematic review and meta-analysis. Hum Reprod Update 12: 651–671.
3. Al-Inany HG, Youssef MAFM, Aboulghar M, Broekmans FJ, Sterenburg MD, et al. (2011) Gonadotrophin-releasing hormone antagonists for assisted reproductive technology. Cochrane Database Syst Rev 11: CD001750.
4. Depalo R, Jayakristan K, Garrutu G (2012) GnRH agnist versus GnRH antagonist in in vitro fertilization and embryo transfer (IVF/ET). RBMO 10: 26.
5. Al-Inany HG, Youssef MAFM, Aboulghar M, Broekmans FJ, Sterenburg MD, et al. (2011) Gonadotrophin-releasing hormone antagonists for assisted reproductive technology. Cochrane Database Syst Rev 11: CD001750.
6. Humaidan P, Kol S, Papanikolaou EG (2001) Copenhagen GnRH agonist triggering workshop group: GnRH agonist for triggering of final oocyte maturation: Time for a change of practice? Hum Reprod Update 4: 510-524.
7. Devreker F, Pogonici E, Revelard P, Bergh M, Englert Y (1999) Selection of good embryos for transfer depends on embryo cohort size: Implications of “mild ovarian stimulation” debate. Human Reprod 14: 3002-3008.
8. Opsahl MS, Blauer KL, Black SH, Lincoln SR, Thorsell LL, et al. (2001) The number of embryo available for transfer predicts successful pregnancy outcome in women over 39 years with normal ovarian reserve testing. J Assit Reprod Genet 18: 551-556.
9. Klein N, Houmard BS, Sluss PM, Sloules MR (2002) Is the short follicular phase in older women secondary to advanced or accelerated dominant follicle development? J Clin Endocrinol Metab 87: 5746-5750.
10. Fanchin R, Salomon L (2003) Luteal oestradiol pre-treatment coordinates follicular growth during controlled ovarian hyperstimulation with GnRH antagonists. Human Reprod 18: 2698-2703.
11. Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E et al. (1996) Hormonal regulation of apoptosis in early antral follicles: Follicle-stimulating hormone as a major survival factor. Endocrinology 137: 1447-1456.
12. Kolibianakis EM, Venetis CA, Bontis J, Tarlatzis BC (2012). Significantly lower pregnancy rates in the presence of progesterone elevation in patients treated with Gnrh antagonists and gonadotrophins: A systematic review and meta- analysis. Curr Pharm Biotechnol 13: 464-470.
13. Venetis CA, Kolibianakis EM, Bosdou JK, Tarlatzis BC (2013) Progesterone elevation and probability of pregnancy after IVF: A systematic review and meta-analysis of over 60 000 cycles. Hum Reprod Update 19: 433-457.
14. Bosch E, Valencia W, Escudero E, Crespo J, Simon C, et al. (2003) Premature luteinization during gonadotropin-releasing hormone antagonist cycles and its relationship with in vitro fertilization outcome. Fertil Steril 80: 1444-1449.
15. Bosch E, Labarta E, Crespo J, Simon C, Remohi J, et al. (2010) Circulating progesterone levels and on-going pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: Analysis of over 4000 cycles. Hum Reprod 25: 2092-2100.
16. Van Vaerenbergh I, Fatemi HM, Blockeel C, Van Lommel L, In't Veld P, et al. (2001) Progesterone rise on HCG day in Gnrh antagonist/RFSH stimulated cycles affects endometrial gene expression. Reprod Biomed Online 22: 263-271.
17. Xu B, Li Z, Zhang H, Jin L, Li Y, et al. (2012) Serum progesterone level effects on the outcome of in vitro fertilization in patients with different ovarian response: An analysis of more than 10,000 cycles. Fertil Steril 97: 1321-1327.
18. Kyrou D, Al-Azemi M, Papanikolaou EG, Donoso P, Tziomalos K, et al. (2012). The relationship of premature progesterone rise with serum estradiol levels and number of follicles in Gnrh antagonist/recombinant FSH-stimulated cycles. Eur J Obstet Gynecol Reprod Biol 162: 165-168.
19. Huang CC, Lien YR, Chen HF, Chen MJ, Shieh CJ, et al. (2012). The duration of pre-ovulatory serum progesterone elevation before HCG administration affects the outcome of IVF/ICSI cycles. Hum Reprod 27: 2036-2045.