Abstract
Purpose
To compare the effectivity of flexible-start medroxyprogesterone acetate (MPA) co-treatment ovarian stimulations (OS) with flexible-start gonadotropin-releasing hormone antagonist (GnRH-ant) co-treatment OS, in blastocyst freeze-all IVF cycles.
Method
This matched cohort study was performed at a single IVF center. Study cycles were extracted from freeze-all IVF cycles performed between February 2015 and June 2018 with cycles grouped according to the co-treatment protocol (MPA and GnRH-ant groups) used. MPA cycles were matched 1:1 using antral follicle count, female age, infertility duration, and female body mass index, with GnRH-ant cycles, resulting in 825 matched cycles. MPA or CET co-treatment was started when leading follicles reached 11–12 mm.
Results
Duration of OS was significantly longer, and total FSH dose was significantly higher in the MPA group. Numbers of mature oocytes retrieved were similar; however, the mature oocyte retrieval rate (83.8 vs. 97.1%; p < 0.001), number of blastocysts, blastocyst rate (36.4 vs. 41.4%; p < 0.001) and > 2 viable blastocyst rate were all significantly lower in the MPA group. The live birth (LB) per transfer rates (51.6 vs. 55.7%; p = 0.155) were similar; however, the LB rate per treatment was significantly lower (40.9 vs. 45.8%; p = 0.05). A linear regression included the OS co-treatment protocol (GnRH-ant; 1.4 (1.07-1.81); p = 0.013) in the final model to predict having > 2 viable blastocysts.
Conclusion
Flexible-start MPA co-treatment OS was as effective in freeze-all IVF cycles as GnRH-ant co-treatment, with similar LB per transfer rates; however, increased cycle cancellation and reduced blastocyst numbers reduced LB per treatment rates significantly.
Keywords: Medroxyprogesterone acetate, Flexible start, Co-treatment, Ovarian stimulation, Freeze-all IVF
Introduction
Advances in vitrification technology have led to improved preservation of embryo quality and, therefore, to increased frozen embryo transfer (FET) implantation rates that were now equivalent to those of fresh ET [1]. The improvement in the clinical outcomes of FET has seen confidence in the use of freeze-all IVF treatment increase, which has now encouraged investigating the introduction of novel ovarian stimulation (OS) protocols in IVF treatment. The success of IVF has always been contingent upon the number of oocytes retrieved from OS cycles [2–4]. While the serial administration of high doses of exogenous gonadotropins has the potential to stimulate multi-follicle development [5], the increased plasma levels of progesterone and estrogen associated with high gonadotropin dose multi-follicle developments may prematurely initiate mid-cycle luteinizing hormone (LH) surges, resulting in cycle cancellation or compromise treatment outcomes [6]. Multi-follicle OS in IVF, therefore, requires pituitary suppression of gonadotropin-releasing hormone (GnRH) to prevent premature LH surges, which has conventionally been achieved using gonadotropin-releasing hormone (GnRH) analogue (i.e., agonist and antagonist) co-treatments [7].
While bioactivities of available GnRH analogues make them effective in support of the multi-follicular IVF strategy, their use increases OS management complexity and the risks of life-threatening side effects such as ovarian hyperstimulation syndrome (OHSS) [5, 7]. Even though OS protocols were introduced that included gonadotropin stimulation, GnRH antagonist co-treatment, and GnRH agonist ovulation trigger, which used in conjunction with freeze-all IVF treatment reduced OHSS significantly [8], the management complexity and costs remained high. Reproductive endocrinologists have for long known that progestin (i.e., artificial progesterone) could be a feasible alternative to conventional GnRH-analogue co-treatment in OS for IVF, because of the long-standing evidence that progestin formulations in oral contraceptive therapies effectively prevent LH surges and ovulation [9]. The presumed risk to normal embryo-endometrial interaction in IVF with fresh ET [10], however, prevented low-cost progestin-based LH suppression protocols from being introduced in routine IVF. The recent evolvement of effective freeze-all IVF in which high mid-cycle progesterone levels is not of concern because of embryo transfer being postponed [11], however, has now made it possible to investigate the efficacy of progestin co-treatment OS in IVF.
Patient-friendly progestin co-treatment OS or progestin-primed ovarian stimulation (PPOS), as it has become known, was first investigated in follicular phase OS in the randomized controlled trial (RCT) of Kuang et al. [12]. The RCT was performed after the authors observed that endogenous luteal progesterone levels effectively suppressed LH in luteal phase OS, without compromising the developmental competence of resultant oocytes and embryos [13]. In this first RCT, the reproductive outcomes of good prognosis patients who had undergone medroxyprogesterone acetate (MPA) co-treatment OS were found to be similar to those of patients who had undergone conventional short-protocol GnRH agonist co-treatment OS. Since then, progestin co-treatment in follicular phase OS was shown to effectively prevent premature LH surges in the OS of different patient populations: diminished ovarian reserve [14], polycystic ovarian syndrome [15, 16], and high body mass index (BMI) [17] patients. Subsequent studies have also shown that different types of progestin were equally effective in preventing LH surges, including Utrogestan [18] and dydrogesterone [19]. Nonetheless, with its moderate bioactivity and low androgenicity, MPA has become the progestin most used in studies.
MPA co-treatment OS was chosen to be introduced at the IVF center based on published and anecdotal evidence, to increase flexibility in OS for IVF and to reduce the financial costs and psychological burden on patients. In the present study, the effectiveness of MPA to support mid-cycle folliculogenesis, ovulation, and oocyte retrieval was investigated, by comparing the reproductive outcomes of flexible-start MPA co-treatment with GnRH-ant co-treatment OS cycles in non-elective blastocyst freeze-all IVF.
Materials and methods
Patients
In this matched cohort study performed at a single IVF center, treatment cycles of patients registered with the clinicians of the IVF center were extracted from freeze-all IVF cycles performed between February 2015 and June 2018. The study was performed in compliance with the regulatory requirements and approval of the Clinical Research Ethics Committee of the Medical Faculty of Akdeniz University (Ref. #1181: 11.12.2019), with all patients providing informed consent before starting treatment. At the IVF center, all IVF treatments (> 95%) were performed as freeze-all IVF cycles, using only autologous gametes, intracytoplasmic sperm injection (ICSI), blastocyst vitrification, and artificial cycle FET. Treatment cycles, in which female age was > 42 years, consent to anonymized data use in research was not given, preimplantation genetic testing (PGT) was performed, and in which fresh ET was performed, were all excluded. The remaining treatment cycles were screened to only include a patient’s first treatment cycle and grouped according to the type of flexible-start co-treatment OS protocol used: MPA co-treatment (MPA group) and GnRH antagonist co-treatment (GnRH-ant group). Treatment cycles from the two groups were matched 1:1, using patient variables in the following order of priority antral follicle count (AFC) (1), female age (2), infertility duration (3), and BMI (4), with no patient included in more than one matching and only the best matched treatment cycles included in the final analysis.
Procedures
Ovarian stimulation and final oocyte maturation
TVS antral follicle assessments were performed on days 2–3 of treatment cycles. AFC, female age, body mass index (BMI), infertility duration, and infertility etiology were included in algorithms to determine starting doses of recombinant follicle-stimulating hormone (rFSH, 150-375 IU, Gonal-F, Merck Serono, Istanbul, Turkey) and human menopausal gonadotropin (hMG, 75-150 IU, Menopur, Ferring Pharmaceuticals, Istanbul, Turkey). All patients started rFSH and hMG, administration on cycle days 2–3, with follicular growth assessed on the 5th day of gonadotropins and, thereafter, assessed every 2–3 days. The dose of gonadotropins administered was adjusted according to follicular growth (i.e., follicle size). MPA (2 × 5 mg tabs bid, Tarlusal, Deva Holding A.Ş., Istanbul Turkey) or GnRH-ant (1×, 0.25 mg Cetrotide, Merck Serono, Istanbul, Turkey) co-treatment was started when leading follicles reached 11–12 mm and continued to the day of final oocyte maturation trigger. Final oocyte maturation was triggered with either GnRH agonist (2× dose, 0.1 mg Gonapeptyl®, Ferring Pharmaceuticals, Istanbul, Turkey) or a combination of GnRH agonist (2× dose, 0.1 mg) and human chorionic gonadotropin (hCG, 250 μg , Ovidrel, Merck Serono, Istanbul, Turkey) as soon as ≥ 3 follicles reached ≥ 17 mm. The decision to use MPA co-treatment was made by patients before the start of treatment in consultation with their clinician, with the decision based only on cost and psychological factors. The decision on which trigger type to administer was made by the patient’s clinician, with the decision based on previous OS outcomes and current follicle size dispersion.
Oocyte retrieval and blastocyst development
Transvaginal follicular aspirations were performed under general anesthesia 36 h after ovulation was triggered. Oocyte-cumulus complexes (OCCs) were retrieved from follicular aspirates, with ICSI performed on all mature (i.e., metaphase II) oocytes 40 h after trigger. In vitro embryo cultures were performed using single-step culture medium protocols, with in vitro culture conditions set at 6% CO2, 5% O2, and 37.0 °C. Embryos developing from normally fertilized zygotes (2-pronuclear zygotes) were assessed daily [20], with blastocysts scored and selected on post-injection days 4, 5, and 6 for cryopreservation [21]. All viable blastocysts were cryopreserved using vitrification protocols and technologies, as specified by the manufacturer (Cryotop, Kitazato BioPharma Co. Ltd, Fuji City, Japan).
Artificial frozen embryo transfer cycles
FETs were performed in programmed artificial cycles [22]. Endometria were prepared using step-up oral estrogen (2 mg, Estrofem, Novo Nordisk, Istanbul, Turkey) protocols. On day 14 of estrogen administration, endometrial thickness was assessed and serum progesterone level measured, with cycles cancelled according to the arbitrary thresholds of 7.0 mm and 1.5 ng/mL, respectively. Progesterone (90 mg, Crinone® 8%, Merck Serono, Istanbul, Turkey) administration was started on the morning (am) of day 15, with the start date of progesterone and the day of blastocyst cryopreservation used to coordinate the day of FET [22]. Blastocysts cryopreserved on post-injection days 5 and 6 were transferred on day 6, and blastocysts cryopreserved on day 4 were transferred on day 5 of progesterone administration [21]. All blastocysts were transferred under trans-abdominal ultrasound guidance, with a maximum of two blastocysts transferred. Hormone supplementation was continued in pregnant patients until 10 weeks of gestation.
Outcomes and statistical analysis
In the present study, the primary outcome measure was mature oocyte retrieval, measured as the number of mature oocytes retrieved and mature oocyte retrieval rate. The secondary outcome measures were duration of OS, number of viable blastocysts, and live birth (LB) from only the first FET cycles following blastocyst freeze-all. The mature oocyte retrieval rate was defined by the ratio: number of mature oocytes retrieved/number of ≥ 14 mm follicles observed on the day of trigger. Cycle cancellation was defined as a cycle in which no viable blastocyst was available for cryopreservation, as the result of zero oocytes, zero mature oocytes, zero fertilized zygotes, or total embryo developmental arrest. A blastocyst was defined as an embryo with a blastocoel that allowed the morphological assessment of the inner cell mass (ICM) and trophectoderm (TE) [23]. Serum LH levels (mIU/mL) were measured on the day of final oocyte maturation trigger (i.e., day of trigger) in only the progestin group, with a premature LH surge defined as a LH level of ≥ 15.0 mIU/mL LH level and severe pituitary suppression defined as a LH level of < 1 mIU/mL. A LB was defined as the delivery of a live infant at > 20 weeks of gestation [24]. The LB rate per treatment was defined by the ratio: number of live births following one FET/number of started treatments. A clinical pregnancy was defined as a pregnancy cycle with normal fetal heart activity confirmed by ultrasound after 5 weeks of gestation. A pregnancy cycle was defined as a cycle in which an arbitrary βHCG level of > 30 mIU/mL was measured 9 days after blastocyst transfer. Implantation rate was defined by the ratio: number fetal hearts observed/number of blastocysts transferred. An antral follicle was defined as an ovarian follicle observed on ultrasound with a diameter of > 2 and ≤ 10 mm on ovarian cycle days 2–3, with patients having AFC of ≤ 5 diagnosed as having decreased ovarian reserve (DOR). A mature follicle was defined as a follicle with a diameter of ≥ 14 mm on the day of trigger.
SPSS 11.5 (Statistical Package for Social Science version 11.5) was used in all statistical analyses, with variables analyzed as means (plus standard deviations), medians (plus interquartile ranges 25% and 75%), or as rates (percentage ratios). Mann-Whitney rank sum tests and chi-square tests (or Fisher exact test in the case of low cycle numbers) were performed in univariate comparative analyses, with significant difference indicated by a p < 0.05. A sample size calculation was performed to indicate the minimum sample size required in a matched cohort study to detect a change in mature oocyte retrieval rate of 15%, at a power of 80% and a type-I error probability of 0.05. The mature oocyte retrieval rate in the control cohort was expected to be 90% based on experience, which meant that at least 128 freeze-all IVF cycles had to be included in each cohort. A forward stepwise (conditional) linear regression was performed to predict an arbitrary number (> 2) of viable blastocysts, with the odds ratios (OR) and 95% confidence intervals (CI) reported for the significant (p < 0.05) variables selected. In a sub-group analysis, outcomes were also tabulated according to AFC sub-groups: 1–5, 6–15, and > 15. The patient and treatment variables included in the regression were selected based on the model of a previously performed multiple logistic regression, performed to predict blastocyst freeze-all [25]. In the forward stepwise linear regression of the present study, the following list of independent variables were included: female age, infertility duration, AFC, BMI (kg/m2), duration of OS, total FSH dose, final maturation trigger type, mature oocyte retrieval rate, number of mature oocytes, oocyte maturation rate, number of 2PN zygotes, fertilization rate, and OS co-treatment protocol.
Results
In this matched cohort study, treatment cycles included in the analysis were extracted from the freeze-all IVF cycles performed during the study period, with a female age of 32.5 ± 5.28 (18-42) and an annual pregnancy rate of 68.2 ± 1.65%. The treatment cycles extracted were grouped according to the co-treatment protocol used in OS, i.e., flexible-start MPA and GnRH-ant co-treatments. MPA co-treatment cycles were matched 1:1 with GnRH-ant co-treatment cycles, resulting in 825 matched treatment cycles (Table 1). The proportions of treatment cycles with AFC 1–5 (16.8%), 6–15 (49.8%), and > 15 (33.5%) were identical (p = 1.000) in the two groups, with AFC ranging from 1 to 99. Female age, infertility duration, and female BMI in the matched groups were statistically similar, with the distribution of female age (≤ 25, 26–30, 31–35, and ≥ 36 years) group cycles also similar. The proportions of major (> 76% of cycles) patient infertility etiologies (unexplained, male, and DOR) cycles were also statistically similar (Table 1).
Table 1.
Patient demographics and infertility etiologies
| MPA group N = 825 |
GnRH-ant group N = 825 |
p value | |
|---|---|---|---|
| Female age (years) | 32.8 ± 4.98 | 32.7 ± 5.11 | |
| 33.2 (29.0–36.7) | 33.1 (28.8–36.5) | = 0.741 | |
| ≤ 25 | 8.2 (68) | 9.2 (76) | = 0.541 |
| 26–30 | 25.2 (208) | 24.8 (205) | = 0.910 |
| 31–35 | 32.7 (270) | 33.8 (279) | = 0.676 |
| ≥ 36 | 33.8 (279) | 32.1 (265) | = 0.496 |
| Antral follicle count (n) | 14.2 ± 10.90 | 14.2 ± 10.88 | |
| 12.0 (7.0–18.0) | 12.0 (7.0–18.0) | = 1.000 | |
| Infertility duration (years) | 4.0 (2.0–7.0) | 4.0 (2.0–7.0) | = 0.257 |
| Body mass index (kg/m2) | 25.0 (22.0–28.0) | 25.0 (23.0–28.0) | = 0.194 |
| Primary infertility etiologies | |||
| Unexplained | 31.0 (256) | 27.4 (226) | = 0.116 |
| Male | 26.3 (217) | 27.3 (225) | = 0.697 |
| Decreased ovarian reserve | 25.8 (213) | 22.2 (183) | = 0.095 |
| Anovulation | 5.0 (41) | 5.9 (49) | = 0.448 |
| Endometriosis | 1.0 (8) | 0.8 (7) | = 1.000 |
| Tubal | 6.3 (52) | 9.3 (77) | = 0.028 |
| Other | 4.6 (38) | 7.0 (58) | = 0.046 |
Data presented as mean ± standard deviation, median (interquartile range), or percentage (number), and statistical significance (p < 0.05). GnRH-ant group includes cetrorelix acetate (Cetrotide, Merck Serono, Istanbul, Turkey) ovarian stimulation cycles. Patient cycles matched according to antral follicle count > female age > infertility duration > body mass index
In flexible-start MPA co-treatment OS, the duration of OS was significantly longer and the total dose of FSH administered significantly higher than in GnRH-ant co-treatment OS (Table 2). Even though the numbers of mature follicles triggered were significantly higher in the MPA group, the numbers of mature oocytes retrieved were similar in the two groups, which resulted in the mature oocyte retrieval rate being significantly lower in the MPA group. The maturation rates of oocytes retrieved were similar in the two groups, with the in vitro embryo development procedures performed resulting in fertilization rates that were not significantly different; however, the number of blastocysts, blastocyst rate, and > 2 viable blastocyst rate were all significantly lower in the MPA group. These clinical and laboratory outcomes were observed to be moderated by the AFC of patients. In treatment cycles with AFC 6–15, the durations of OS (10 (9–11) vs. 9 (8–10); p < 0.001), mature oocyte retrieval rates (75.0 (58.6–100.0) vs. 100.0 (73.3–125.0); p < 0.001), and blastocyst rates (36.4 (16.7–50.0) vs. 42.9 (25.0–64.3); p < 0.001) were similarly significantly different in the two groups. On the contrary, in treatment cycles with AFC > 15, the durations of OS (9 (8–10) vs. 9 (9, 10); p = 0.293), mature oocyte retrieval rates (92.9 (73.3–112.5) vs. 95.8 (75.0–125.0); p = 0.071), and blastocyst rates (37.5 (25.0–50.0) vs. 40.0 (28.6–52.2); p = 0.104) were all statistically similar in the two groups. In a forward step-wise (conditional) linear regression to predict having > 2 viable blastocysts in a treatment cycle (Table 3), the following variables were selected as significant independent predictors in the final step (step 4): duration of OS (p < 0.001), total FSH dose (p = 0.011), number of 2PN zygotes (p < 0.001), and OS co-treatment protocol (GnRH-ant; p = 0.013).
Table 2.
Treatment outcomes according to the co-treatment protocol used in OS
| MPA group N = 825 |
GnRH-ant group N = 825 |
p value | |
|---|---|---|---|
| Duration of ovarian stimulation (days) | 9.7 ± 1.78 | 9.0 ± 1.74 | |
| 10 (9.0–11.0) | 9 (8.0–10.0) | < 0.001 | |
| Total FSH dose (IU) | 4050 (3000–4500) | 3600 (2700–4050) | < 0.001 |
| Mature follicle number (n) | 11.3 ± 6.85 | 10.7 ± 7.07 | |
| 10 (6.0–16.0) | 9 (5.0–15.0) | = 0.034 | |
| Mature oocyte number (n) | 9.8 ± 7.69 | 10.3 ± 7.73 | |
| 8 (4.0–14.0) | 9 (4.0–14.0) | = 0.185 | |
| Mature oocyte retrieval rate (%) | 83.8 ± 39.7 | 97.1 ± 43.0 | |
| 82.4 (60.0–100.0) | 100.0 (71.4–121.8) | < 0.001 | |
| Mature oocyte rate (%) | 80.0 (66.7–92.6) | 80.0 (66.7–91.7) | = 0.735 |
| Fertilization rate (%) | 81.8 (66.7–100.0) | 81.8 (66.7–94.9) | = 0.919 |
| Blastocyst rate (%) | 36.4 (20.0–50.0) | 41.4 (25.0–60.0) | < 0.001 |
| Blastocyst number (n) | 2.8 ± 2.74 | 3.3 ± 2.91 | |
| 2 (1.0–4.0) | 3 (1.0–5.0) | < 0.001 | |
| > 2 viable blastocyst rate (%) | 43.6 (360) | 51.9 (428) | < 0.001 |
| Cycle cancellation rate (%) | 20.4 (168) | 17.7 (146) | = 0.188 |
| Zero oocytes (%) | 1.3 (11) | 1.7 (14) | |
| Zero mature oocytes (%) | 1.8 (15) | 0.8 (7) | |
| Zero normal fertilization (%) | 1.3 (11) | 1.8 (15) | |
| Embryo development arrest (%) | 15.9 (131) | 13.3 (110) | |
| Blastocyst freeze-all rate (%) | 79.6 (657) | 82.3 (679) | = 0.188 |
| Number of blastocysts transferred (n) | 851 | 936 | |
| 1,33 ± 0.470 | 1.39 ± 0.487 | ||
| 1 (1.0–2.0) | 1 (1.0–2.0) | = 0.026 | |
| Implantation rate (%) | 50.2 (427) | 52.6 (492) | = 0.336 |
| Incomplete cycles (%) | 16 | 4 | |
| FET (n) | 641 | 675 | |
| FET pregnancy rate (%) | 68.8 (441) | 72.4 (489) | = 0.164 |
| FET clinical pregnancy rate (%) | 58.2 (373) | 63.0 (425) | = 0.086 |
| FET live birth rate (%) | 51.6 (331) | 55.7 (376) | = 0.155 |
| Total pregnancy loss rate (%) | 24.9 (110) | 23.1 (113) | = 0.564 |
| Treatment cycles (n) | 809 | 821 | |
| Treatment live birth rate (%) | 40.9 (331) | 45.8 (376) | = 0.05 |
Data presented as mean ± standard deviation, median (interquartile range), or percentage (number), and statistical significance (p < 0.05). FSH: follicle-stimulating hormone; mature follicle: ≥ 14 mm follicle on the day of trigger; mature oocyte: metaphase II oocyte; normal fertilization: 2PN zygote; blastocyst: a viable blastocyst suitable for cryopreservation; implantation rate: number of fetal hearts/number of blastocysts transferred; incomplete cycles: no ET within the study period; treatment cycles: completed treatments; treatment live birth rate: live birth rate per started treatment (intention-to-treat)
Table 3.
Forward conditional logistic regressions to > 2 viable blastocysts
| Variables selected | OR (95% CI) | p value | B ± SE |
|---|---|---|---|
| Duration of ovarian stimulation | 0.81 (0.716–0.904) | < 0.001 | − 0.22 ± 0.06 |
| total FSH dose | 1.00 (1.0–1.0) | = 0.011 | 0.00 ± 0.00 |
| number of 2PN zygotes | 1.48 (1.42–1.54) | < 0.001 | 0.39 ± 0.02 |
| OS co-treatment protocol | 1.40 (1.07–1.81) | = 0.013 | 0.33 ± 0.13 |
| constant | 0.15 | < 0.001 | − 1.890 ± 0.398 |
Data presented as odds ratios (OR and 95% confidence interval), regression coefficient and standard error (B ± SE), and statistical significance (p < 0.05). The following variables were included: dependent variable > 2 viable blastocysts (0 ≤ 2 or 1 > 2), independent variables of female age, infertility duration, AFC, BMI, duration of OS, total FSH dose, final maturation trigger type, mature oocyte retrieval rate, number of mature oocytes, oocyte maturation rate, number of 2PN zygotes, fertilization rate, and OS co-treatment protocol
The cycle cancellation rates were similar in the MPA and GnRH-ant groups (Table 2), with similar distributions across the cancellation categories shown in Table 2. Six hundred and fifty-seven (79.6%) of the 825 treatment cycles in the MPA group resulted in blastocyst freeze-all, which was similar to the rate (82.3%) in the GnRH-ant group. In both the MPA (n = 16) and GnRH-ant (n = 4) group, there were incomplete cycles (i.e., cycles with no FET within the study period). The numbers of blastocysts transferred in the MPA group were significantly lower than that in the GnRH-ant group, with 431 (67.2%) single blastocyst transfers performed in the MPA group and 414 (61.3%) in the GnRH-ant group (Table 2). The implantation rates from all FET performed were statistically similar (50.2% (427/851) vs. 52.6% (492/936); p = 0.336), as were the pregnancy rates from single blastocyst transfers (64.0% (276/431) vs. 67.9% (281/414); p = 0.270). The pregnancy, clinical pregnancy and LB rates from all the FET (i.e., per FET rates) performed in the two groups were also all statistically similar, as well as the total pregnancy loss rates (Table 2). The LB rate from the 825 freeze-all IVF treatments (i.e., per treatment rate, including cancelled cycles and excluding incomplete cycles) performed in the MPA group was significantly lower than the rate in the GnRH-ant group (40.9 vs. 45.8%; p = 0.05; Table 2).
In the MPA group, serum LH levels were measured on the day of trigger in all cycles. Of LH, ≥ 15 mIU/mL was the threshold level used to define a premature LH surge in the present study. Of the 825 treatment cycles in the MPA group, 24 (2.91%) had serum LH levels of ≥ 15 mIU/mL and 77 (9.3%) had severe LH suppression levels (< 1.0 mIU/mL). The mean day-of-trigger LH level in the MPA group was 4.97 ± 3.901. In the GnRH-ant group, a limited number of treatment cycles (n = 450) had LH levels measured on the day of trigger, with a mean day-of-trigger LH level of 2.65 ± 3.184 mIU/mL. The effect of ≥ 15 mIU/mL LH levels on the treatment outcomes of the MPA group is presented in Table 3. Demographically, patients in the ≥ 15 mIU/mL sub-group were significantly older and had significantly lower AFC. The number of mature oocytes retrieved was significantly lower in the ≥ 15 mIU/mL sub-group. The pregnancy rate of the ≥ 15 mIU/mL sub-group was not significantly different to that of the < 15 mIU/mL sub-group because of the low numbers of FET performed in the < 15 mIU/mL sub-group. Moreover, the pregnancy rate of the sub-group with < 1.0 mIU/mL LH levels (72.3%; 48/66) was statistically similar to that of the GnRH-ant group (72.4%; 489/675).
Discussion
Recent studies have shown that progestin co-treatment could effectively prevent premature LH surges during OS for IVF, without detrimentally affecting mid-cycle folliculogenesis and pre- and post-implantation embryogenesis outcomes (Table 4) [6, 10]. These studies, therefore, prompted the suggestion that progestin co-treatment could be a feasible alternative for conventional GnRH-analogue co-treatment OS in IVF requiring a freeze-all (i.e., fertility preservation, oocyte/embryo donation, PGT, luteal phase OS, and elective freeze-all IVF).
Table 4.
Progestin co-treatment OS cycles with LH levels of ≥ 15 mIU/mL
| < 15 mIU/mL | ≥ 15 mIU/mL | p value | |
|---|---|---|---|
| Treatments | 801 | 24 | |
| Female age (years) | 32.7 ± 4.99 | 35.4 ± 4.26 | = 0.012 |
| 33.1 (28.9–36.6) | 34.6 (32.4–39.3) | ||
| Antral follicle count (n) | 12 (7.0–18.0) | 6.5 (3.0–12.5) | = 0.002 |
| Mature oocyte number (n) | 8 (4.0–14.0) | 3.5 (1.0–8.0) | < 0.001 |
| Mature oocyte retrieval rate (%) | 83.3 (60.0–101.7) | 74.2 (33.3–100.0) | = 0.212 |
| Blastocyst rate (%) | 36.4 (20.0–50.0) | 33.3 (0.0–57.1) | = 0.574 |
| Blastocyst freeze-all rate (%) | 80.5 (645/801) | 50.0 (12/24) | = 1.0001 |
| Pregnancy rate (%) | 69.0 (435/630) | 54.5 (6/11) | = 1.0001 |
Data presented as mean ± standard deviation, median (interquartile range), or percentage (number), and statistical significance (p < 0.05)
1Fisher exact test
In the present study, the clinical effectivity of MPA co-treatment OS was investigated in a matched cohort study of non-elective freeze-all IVF cycles, matching flexible-start MPA co-treatment cycles 1:1 with flexible-start GnRH-ant co-treatment cycles. Premature LH surges (≥ 15 mIU/mL) occurred in 24 (2.91%) treatment cycles of the MPA group; notwithstanding, the cancellation rate of the MPA group was similar (20.4 vs. 17.7%; p = 0.188) to that of the GnRH-ant group. While similar pregnancy, clinical pregnancy, implantation, and LB rates indicated that MPA co-treatment had no adverse effect on the developmental competence of blastocysts transferred, the significantly lower mature oocyte retrieval, blastocyst, and > 2 viable blastocyst rates suggest that MPA co-treatment may have some effect on mid-cycle folliculogenesis and, therefore, also on pre-implantation embryogenesis. In a linear regression, the co-treatment protocol used was included in the final model as a significant independent variable predicting > 2 viable blastocysts (p = 0.013). The evidence of the present study, therefore, suggests that MPA co-treatment reduces per treatment LB (i.e., intention-to-treat) and potentially may also reduce cumulative reproductive potential of IVF treatment. Moreover, a longer duration of OS and a higher total FSH dose was confirmed in MPA co-treatments, notwithstanding the use of flexible-start protocols (i.e., MPA co-treatment was only started after leading follicles reached 11–12 mm). Interestingly, the duration of OS and total FSH dose were also selected in the linear regression model as significant independent predictors of > 2 viable blastocysts.
Even though the MPA group had a significantly higher number of mature follicles (10 (6.0–16.0) vs. 9 (5.0–15.0); p = 0.034) and a relatively high mature oocyte retrieval rate (83.8% ± 39.7), the numbers of mature oocytes retrieved were similar in the two groups (8 (4.0–14.0) vs. 9 (4.0–14.0); p = 0.185). The reason being that the high mature oocyte retrieval rate in the MPA group was still significantly lower than that in the GnRH-ant group. This lower oocyte retrieval rate was not noted in clinicians’ oocyte retrieval reports, to be the result of premature ovulation. In a previous study [26], 2.7% of GnRH agonist triggers resulted in sub-optimal responders, with sub-optimal responders having a significantly lower oocyte retrieval rate (48.16 vs. 68.26%). In the present study, GnRH agonist triggers were administered in 98.7% (814/825) of MPA co-treatments, with neither mature oocyte retrieval rate nor trigger type selected as independent predictors of > 2 viable blastocysts. Moreover, exploratory and presented analyses of the data showed that both LH level and AFC were important covariables in MPA co-treatments, with mature oocyte retrieval rates decreasing with increasing LH levels and decreasing AFC. The mean mature oocyte retrieval rate was the lowest in treatment cycles with LH levels ≥ 15 mIU/mL; however, in this sub-group, female age was increased and AFC decreased. The mid-cycle administration of high doses of MPA may increase the risk of LH-related dysfunctional ovulation (i.e., retarded oocyte maturation or incomplete maturation and expansion of cumulus cells), which has been suggested to be potential reasons for failing to retrieve all oocytes (i.e., the failure of OOC to be released and the failure to identify immature OCC) [27].
The majority of previous studies, in which progestin co-treatment OS were investigated, followed the fixed-start protocol proposed in the seminal study of Kuang et al. [12]. In this protocol, hMG and MPA administration was started simultaneously on day 3 of an ovarian cycle and continued to the day of trigger. Progestin administration has been shown not to result in the immediate suppression of LH secretion, but rather in the progressive suppression of LH. LH levels on the day of trigger, therefore, were observed to be significantly lower than that on the day progestin co-treatment was started [18, 28, 29]. Moreover, previous studies have consistently reported that OS durations and total gonadotropin doses administered were increased in progestin co-treatment OS [6, 10], the cause and implications of which still remain uncertain. In the study of Kuang et al. [12], 0.7% (1/150) of patients underwent premature LH surges, with a mean LH level of 1.8 ± 1.3 mIU/mL observed on the day of trigger. Low levels of LH on the day of trigger have since been confirmed in other studies, with almost all reporting no premature LH surges [18, 28, 29]. In the present study, flexible-start high-dose MPA co-treatments resulted in LH levels of 4.97 ± 3.90 mIU/mL on the day of trigger, with 92.8% (785/846) of patients having LH levels < 10 mIU/mL on the day of trigger. Fixed-start progestin co-treatment may also increase the risk of severe LH suppression (LH < 1.0 mIU/mL). In the present study, the incidence (9.3%) of severe pituitary suppression in MPA treatments was comparatively low (< 14–28%) [30]. In standard IVF, GnRH-ant co-treatment cycles with excessive as well as with insufficient LH suppression were reported to have reduced reproductive success [31]. In the present study, MPA co-treatment cycles with severely suppressed LH levels were associated with the highest pregnancy rate and MPA co-treatment cycles with LH levels of ≥ 15 mIU/mL were associated with the lowest pregnancy rate.
Ovarian stimulation durations and total gonadotropin doses were reported not to be significantly different in two recent donor-oocyte studies that also investigated MPA co-treatment using flexible-start protocols [29, 32]. This was contrary to the outcomes found in previous studies and those of the present study, in which the duration of OS was significantly longer and total gonadotropin dose administered significantly higher [12]. Importantly, both of these variables were selected as independent predictors of > 2 viable blastocysts in the linear multivariate regression of the present study. In one of these donor-oocytes studies [32], reproductive outcomes (i.e., pregnancy, clinical pregnancy, and LB rates) were nonetheless also not significantly different between the study and control groups, as they were in the present study. This was also consistent with the reproductive outcomes reported in most previous studies in which fixed-start protocols were used [12, 14–17]. In the present study, the implantation and single blastocyst transfer pregnancy rates, as well as the total pregnancy loss rates, were not statistically different between the MPA and GnRH-ant groups. This general consistency in reproductive outcomes suggests that the developmental competence of embryos or blastocysts transferred in FET may be unaffected by progestin co-treatments. Importantly, however, in the present study, MPA co-treatments were found to reduce the LB per treatment rate as the result of a higher cancellation rate, which was the result of a higher embryo development arrest rate. In addition, the reduced > 2 viable blastocysts rate observed may potentially reduce the cumulative reproductive potential of a treatment cycle. Moreover, the encouraging clinical outcomes of MPA co-treatments warrant further investigations to be conducted to refine progestin co-treatment OS (i.e., type of gonadotropin, type of progestin, and type of trigger) and patient selection.
The present study adds significantly to the debate on whether progestin co-treatment OS could be introduced in routine IVF treatments requiring freeze-all. While progestin co-treatment has been proposed as a suitable alternative based on the evidence of previous studies [10, 16], the present evidence cautions against the routine implementation of MPA co-treatment OS because even though MPA co-treatment resulted in the development of blastocysts with normal developmental competence and LB potential, the numbers of viable blastocysts were significantly reduced. Importantly, however, the evidence does suggest that MPA co-treatment could be as effective as conventional GnRH-analogue co-treatment in select patient groups (i.e., patients with AFC > 15). The present study’s strength lies in the accurate matching of 825 patient cycles and the range of patient fertility potential (i.e., female age 19–42 and AFC 1–99) included, as the patient selection in previous studies may have concealed the effects of progestin co-treatment. The weaknesses of the present study include the retrospective analysis of patient cycles, the specific progestin co-treatment protocol used (i.e., flexible-start high dose MPA administration), and the elevated levels of LH observed on the day of trigger. The study limitations and clinical relevance of the differences in LB presented suggest that the outcomes presented should be interpreted with caution. In conclusion, flexible-start MPA co-treatment was as effective in blastocyst freeze-all IVF cycles as flexible-start GnRH-ant co-treatment in terms of LB per transfer; however, because of increased cycle cancellation and reduced viable blastocyst numbers, MPA co-treatment OS may reduce LB per treatment and possibly cumulative LB rates. Moreover, because of the promising clinical and laboratory outcomes of MPA co-treatments in select patients, a large cost differential, and a low psychological burden on patients, further studies are warranted to refine OS protocols to limit any adverse effects on mid-cycle folliculogenesis and ovulation.
Compliance with ethical standards
The study was performed in compliance with the regulatory requirements and approval of the Clinical Research Ethics Committee of the Medical Faculty of Akdeniz University (Ref. #1181: 11.12.2019), with all patients providing informed consent before starting treatment.
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Key message
The results suggest that medroxyprogesterone acetate co-treatment ovarian stimulation should be implemented with caution, because mature oocyte retrieval, blastocyst development, and per treatment live birth outcomes may be reduced. While the reproductive competence of viable blastocysts seems unaffected, the number of viable blastocysts available for transfer may be reduced.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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