Outcomes of ovarian stimulation in the follicular and luteal phases of the menstrual cycle in cancer patients

Abstract

Objective

The purpose of the study was to evaluate the effectiveness of controlled ovarian stimulation in different phases of the menstrual cycle in cancer patients seeking for the preservation of reproductive material before gonadotoxic therapy.

Methods

A total of 140 patients with oncological diseases underwent ovarian stimulation in the standard protocol with GnRH antagonists in the follicular phase of the cycle (n = 68) and in the random-start protocol in the luteal phase of the menstrual cycle without prescribing GnRH antagonists (n = 72).

Results

All patients included in the study were comparable in age and AMH level. There were no differences in the mean number of oocytes retrieved in the follicular phase group, and luteal phase group. Similarly, no significant differences were observed in the number of M II oocytes.

Conclusions

The results of the study demonstrate the equal effectiveness of stimulation protocols in different phases of the menstrual cycle, which allows us to develop a personalized approach to the implementation of reproductive function both in cancer patients and in the routine practice of ART.

Keywords:

• Ovarian stimulation
• random-start ovarian stimulation
• fertility preservation

Introduction

Experts from the leading countries of the world consider ensuring the quality of life of cancer patients a priority area of medical science and practice. On the one hand, this is associated with an increase in the number of patients with cancer, especially among children and young people, on the other hand, with the undoubted achievements of oncology, which makes it possible to diagnose the process at early stages, effectively treat the disease, which contributes to a significant increase in survival rates [1–3].

The foregoing served as the basis for the formation of a new interdisciplinary direction in medicine, which is called oncofertility [4]. The main postulate formulated by specialists in oncofertility care is the need to preserve reproductive material for the purpose of delayed childbearing in young patients with cancer before the start of gonadotoxic therapy, which is highlighted in the international documents and clinical guidelines [5].

The area of fertility preservation is constantly developing. Despite this, to date, the only noninvestigational and unequivocally accepted methods for fertility preservation is controlled ovarian stimulation with subsequent cryopreservation of embryos and unfertilized oocytes [5].

The choice of a protocol for ovarian stimulation in cancer patients is based on a balance between the limited time frame before starting gonadotoxic therapy, and the need to obtain a sufficient number of oocytes and embryos for cryopreservation [6]. The flexibility of random-start protocols suggests their advantage in the context of oncofertility. The possibility of obtaining mature oocytes with stimulation on any day of the menstrual cycle has been in a number of studies. Von Wolff and colleagues were among the first to demonstrate that ovarian stimulation can safely and effectively begin in the luteal phase of the menstrual cycle [6,7]. Later researchers publish a controversial studies about effectiveness of random-start protocols. In studies by Li-Hong Wei et al., it has been shown that oocytes obtained in the luteal phase show a high in vitro fertilization rate. There were no differences in the number of obtained oocytes and embryos depending on the phase of the onset of ovarian stimulation. However, pregnancy rates in the luteal phase stimulation group (46.4%) were significantly higher than in the follicular phase stimulation group (25.8% overall; 22.9% in fresh cycles and 29.6%—during cryotransfers) [8].

At the moment, there is a limited amount of reliable information about the outcomes of stimulation of ovarian function in the luteal phase of the menstrual cycle. The purpose of this work was to evaluate the effectiveness of controlled ovarian stimulation despite the phase of menstrual cycle.

Materials and methods

Study population

We performed a prospective observational study at the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after V.I. Kulakov from February 2019 to December 2020. A total of 257 women who had FP consultation prior to neoadjuvant chemotherapy were identified. 140 patients were enrolled for the study according to inclusion criteria. The criteria for inclusion in the studies were: age from 18 to 42 years, AMG not less than 0.75 ng/ml, planned gonadotoxic therapy (chemotherapy, radiation therapy).

The exclusion criteria: extremely reduced ovarian reserve level, recurrent oncological diseases, distant metastases, prior gonadotoxic treatment.

Patients were randomly assigned to one of two groups based on the day of the presentation. The study was approved by the Ethics Committee of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after V. I. Kulakov. Written informed consent was provided by all participants.

Ovarian stimulation

Patients in the follicular phase group (group 1) underwent conventional ovarian stimulation based on the antagonist protocol with rFSH (Gonal-f, Serono) and HMG gonadotropins (Menopur, Ferring). Hormone testing (estradiol, LH, FSH, progesterone) was performed on D2-3 of the menstrual cycle, on D6 of stimulation, and final oocyte maturation trigger injection day.

Daily gonadotropin injections were initiated between D2 and D4 of the cycle. Ovarian response was assessed on D6 of stimulation, and every 2–3 days thereafter. Whenever oocytes were present >12–14 mm, GnRH antagonists (0.25 mg cetrorelix acetate) were administered. During stimulation, ultrasound examination was used to count the number of antral, growing, and preovulatory follicles. Ovulation was triggered by two vials of 0.1 mg triptorelin acetate (Decapeptyl®) when at least one follicle reached ≥18 mm in diameter. Oocyte puncture was scheduled 36 h later. Any mature oocytes (metaphase II) retrieved were subsequently either cryopreserved by vitrification, or fertilized and preserved as frozen embryos.

Patients in the LP (group 2) underwent ovarian stimulation according to the random-start protocol with rFSH (Gonal-f, Serono) and HMG gonadotropins (Menopur, Ferring) (on day 3 after the ovulation, which corresponded to days 16–18 of the cycle). Ovarian response was assessed on D6 of stimulation, and every 2–3 days thereafter. They did not receive additional GnRH antagonist. During stimulation, ultrasound examination was used to count the number of antral, growing, and preovulatory follicles. Ovulation was triggered by two vials of 0.1 mg triptorelin acetate (Decapeptyl®) when at least one follicle reached ≥18 mm in diameter. Oocyte puncture was scheduled 36 h later. Any mature oocytes (metaphase II) retrieved were subsequently either cryopreserved by vitrification, or fertilized and preserved as frozen embryos.

Patients from the both groups affected by estrogen-sensitive malignancy (estrogen receptors positive breast cancer) received letrozole (Femara, Novartis Europharm, Camberley Surrey, UK) to lower estrogen blood levels during ovarian stimulation with gonadotropins.

The number of retrieved oocytes, mature oocyte counts, daily and total gonadotropin doses, and the duration of stimulation were evaluated.

Statistical analysis

The results were processed on a personal computer using Statistical Analysis for Microsoft Excel 2010 and SPSS V22.0. For determining distribution of data before carrying out comparative quantitative data in the studied tested Kolmogorov–Smirnov test or Shapiro–Wilk, depending on the sample size. To describe quantitative features, having a normal distribution, the arithmetic mean (M) and standard deviation (SD). When distributing qualitative features, with distribution different from normal, we used the median (Me) and interquartile range in Me format (Q1–Q3). Qualitative indicators in absolute and relative values (%). In normal view, data distribution methods were used to assess differences in groups parametric statistics (t-test - for unrelated features). When distributing data other than normal, methods of nonparametric statistics were used (Mann–Whitney test - for unrelated signs). All differences were counted statistically significant if p is less than .05.

Results

Nosological entities of oncological diseases in patients referred for retrieval and cryopreservation of reproductive material were analyzed. The results of the analysis are presented in Figure 1. The most common nosological entities were breast cancer (n = 44).

Figure 1. Nosological entities of oncological diseases.

All study patients were comparable in age, BMI, and serum AMH levels. Baseline characteristics of patients in both groups are presented in the Table 1. The mean age in women in groups 1 and 2 was 33.38 (3.73) and 33.3 (5.47) years, respectively; BMI was 23.2 (2.2) and 21.2 (1.6) kg/m², respectively.

There was statistically significant difference in total gonadotropin doses between the two groups. Cycle characteristics in the follicular and luteal phase stimulation groups are presented in the Table 2. A statistically significantly higher dosage of gonadotropins was used in the luteal phase of cycle.

Particular attention was given to gaining insight into steroidogenesis during ovarian stimulation and a functioning corpus luteum, comparison of hormonal parameters between groups are presented in the Table 3.

On the day of the start of stimulation, the FSH level was statistically significantly higher in patients in the follicular phase of the cycle (9.1 (5.7 − 14.2) IU/L, 7.3 (4.8 − 11.4) IU/L, respectively), in comparison with the value of these hormones during stimulation in the luteal phase. This can be explained by a decrease in FSH levels in response to higher levels of estradiol and progesterone secreted by the corpus luteum during the luteal phase of the menstrual cycle.

It is important to note that the LH concentration at all three points of steroidogenesis monitoring was comparable, which was not associated with the levels of another gonadotropin, FSH. Also in none of the cases of controlled ovarian stimulation in the luteal phase of the cycle, we observed premature ovulation associated with premature peaks of LH. This fact can be explained by the protective effect of endogenous progesterone produced by the functionally active corpus luteum upon stimulation in the luteal phase of the cycle. Therefore, with stimulation in the luteal phase of the menstrual cycle, it is possible to dispense with the additional administration of antGnRH to prevent premature peaks of LH.

Analysis of the E2 level showed statistically significantly higher values of the hormone at the beginning of stimulation in the luteal phase of the cycle, which is natural with a functioning corpus luteum.

At the same time, already on the sixth day of stimulation and on the day of administration of the ovulation trigger, the level of estradiol in the second group was statistically significantly lower, which may indicate the ongoing processes of luteolysis and a decrease in the functional activity of the corpus luteum.

The level of progesterone was also significantly higher in the second phase of the menstrual cycle, as at the time of the start of stimulation of ovarian function − 1.2 (0.6 − 1.6) nmol/L and 14.0 (2.7 − 36.3) nmol/l, p < .001, also on the sixth day of stimulation of ovarian function 1.5 (1.2 − 2.4) nmol/l and 7.8 (2.2 − 21.3) nmol/l, respectively (p < .001). However, on the day of administration of the ovulation trigger, no difference in the level of progesterone was found, which confirms the decrease in the functional activity of the corpus luteum.

No difference was found in numbers of retrieved oocyte-cumulus complexes and mature oocytes, even though during a preliminary assessment, fewer follicles were visualized in the LF cycle, which was explained by the presence of a corpus luteum in one of the ovaries. Parameters of oogenesis in patients depending on the phase of the menstrual cycle are presented in the Table 4.

On day 5 of cultivation, embryos of grade ≥3BB were cryopreserved. The number of embryos suitable for cryopreservation was comparable in both groups [4.1 (1.3) and 5.1 (0.2)].

Table 1. Baseline characteristics of patients in both groups.

	Follicularphase (n = 68)	Lutealphase (n = 72)	p
Age, years	33.38 (3.73)	33.3 (5.47)	.255
BMI, kg/m^2	23.2 (2.2)	21.2 (1.6)	.185
AMH, ng/mL	1.5 (1.1–3.2)	1.8 (1.2–3.5)	.196

Table 2. Cycle characteristics in the follicular and luteal phase stimulation groups.

	Follicularphase (n = 68)	Lutealphase (n = 72)	p
Lengthofstimulation (days)	8 (7–10)	9 (6.5–11)	.477
Gonadotropindosage (IU)	2025 (1706–2288)	2400 (2013–2725)	.002

Table 3. Comparison of hormonal parameters between groups.

	Follicular phase (n = 68)	Luteal phase (n = 72)	p-value
Basal LH-1, IU/L	4.5	5.2	.608
	(2.6–7.3)	(1.9–8.3)
Day 6 LH-2, IU/L	2.4	2.8	.739
	(1.7–7.7)	(0.8–6.9)
Day of trigger admisitration LH-3, IU/L	1.8	1.4	.163
	(1.0–3.1)	(0.3–2.8)
Basal FSH,IU/L	9.1	7.3	.018
	(5.7–14.2)	(4.8–11.4)
Basal Estradiol-1, pmol / l	186	332	<.001
	(155–284)	(222–570)
Day 6 Estradiol-2, pmol / l	2765	1827	.010
	(1200–5336)	(936–2988)
Day of trigger admisitration Estradiol-3, pmol / l	7800	5544	.050
	(4139–12943)	(3047–8126)
Basal Progesterone −1, nmol / l	1.2	14.0	<.001
	(0.6–1.6)	(2.7–36.3)
Day 6 Progesterone −2, nmol / l	1.5	7.8	<.001
	(1.2–2.4)	(2.2–21.3)
Day of trigger admisitration Progesterone −3, nmol / l	3.1	3.7	.323
	(1.3–5.7)	(2.0–6.1)

Table 4. Parameters of oogenesis in patients depending on the phase of the menstrual cycle.

	Follicularphase (n = 68)	Lutealphase (n = 72)	p Value
Number of oocytes retrieved per patient (n)	7 (4–16)	10 (7–14)	.236
Total number of oocytes retrieved (n)	715	766
Number of metaphase II oocytes (n)	520 (72.8%)	557 (72.6%)	.521
MI	84 (11.7%)	68 (8.9%)	.042
GV	73 (10.2%)	100 (13.1%)	.052
Deg	38 (5.3%)	41 (5.4%)	.182

In the group of patients stimulated in the follicular phase of the menstrual cycle, a statistically significant greater number of immature oocytes was obtained (84 (11.7%) in the first group and 68 (8.9%) in the second group, p = .042.

There were no significant differences in the results of the embryological stage—the number of fertilized oocytes, the number of blastocysts, as well as an amount of blastocysts of excellent quality.

Discussion

The results of this study confirmed the possibility and effectiveness of ovarian stimulation in the luteal phase of the cycle, which is extremely important for removing the restrictions on treatment in cancer patients. At the same time, the issues of presence of premature peaks of LH and the need to use antGnRH during stimulation in the second phase of the cycle, are debatable. To resolve these contradictions, we investigated the dynamic levels of hormones during stimulation in the luteal phase, comparing them with those during classical conventional ovarian stimulation in the early follicular phase. The FSH level at the start of ovarian stimulation in the luteal phase of the cycle was lower than in the follicular phase (9.1 (5.7 − 14.2) in FF and 7.3 (4.8 − 11.4) in LF, p = .018), which is explained by negative feedback mechanisms and higher levels of sex steroids secreted by the corpus luteum. Apparently, this fact contributes to the ‘natural’ synchronization of follicular growth, which is confirmed by the results of this study.

It is interesting to note the absence of premature LH peaks during stimulation without the addition of antagonists in the luteal phase of the cycle, which is provided by the protective effect of a high level of endogenous progesterone. This fact was also noted in other publications [9,10]. Simultaneously, at the time of the introduction of the ovulation trigger, comparable values of progesterone were recorded in both groups (3.1 (1.3 − 5.7) nmol/L versus 3.7 (2.0 − 6.1) nmol/L, p = .323, respectively). At the same time, the phenomena of a decrease in the functional activity of the corpus luteum were recorded from 6–7 days of the luteal phase, which was manifested by a decrease in the progesterone level (1.5 (1.2 − 2.4) nmol/l versus 7.8 (2.2 − 21.3) nmol/L, respectively, p < .001). This fact, in our opinion, is practically important in solving the problem of prescribing antGnRH during stimulation in the luteal phase. If stimulation begins in the early secretory phase, with an active corpus luteum, then antGnRH can be dispensed with, if the onset of stimulation occurs in the late secretory phase, during the onset of luteolysis, then antGnRH should be introduced into the stimulation protocol, especially with a reduced ovarian reserve.

Therefore, current research evidence on folliculogenesis suggests the possibility of new approaches for controlled ovarian stimulation in ART programs, not only from the point of view of the day of starting ovarian stimulation, but also form completely new protocols for controlled ovarian stimulation.

Disclosure statement

The authors declare that this research is conducted in the absence of any commercial or financial relationship that can be a potential conflict of interest.