Does gonadotropin-releasing hormone agonist cause luteolysis by inducing apoptosis of the human granulosa-luteal cells?

Objectives To evaluates the effect of different modes of final follicular maturation triggering on the degree of apoptosis of granulosa cells (GCs) and the potential effect on progesterone secretion.

Thirty patients undergoing controlled ovarian hyperstimulation for IVF who received hCG, GnRH agonist, or dual trigger for final follicular maturation were included in the study. Granulosa cells were obtained at the time of oocyte retrieval. The proportion of apoptotic cells was evaluated via TUNEL and immunohistochemistry.

The proportion of apoptotic cells was significantly higher in the GnRH agonist–alone group compared to hCG-alone and the dual trigger groups (13.5 ± 1.5% vs. 7.8% ± 1.8 vs. 10.1% ± 2, respectively, P < 0.01). Moreover, the expression of active-caspase-3 was also significantly increased in the GnRH agonist–alone group compared with the hCG-alone and the dual trigger groups (15.5% ± 2.9 vs. 8.4% ± 1.6 vs. 12.7% ± 2.6, respectively, P < 0.01). The progesterone levels measured in the granulosa-luteal cell culture medium after 24 h of incubation were similar between the three groups.

The levels of apoptosis are increased after GnRH agonist/dual trigger. The increased apoptosis might be one of the culprit of the subsequent premature demise of the corpus luteum post GnRH agonist trigger.

GnRH agonist trigger, Dual trigger, Apoptosis

Several studies have shown that the use of gonadotropin-releasing hormone (GnRH) agonist for triggering final follicular maturation prevents the development of ovarian hyperstimulation syndrome (OHSS), even in patients at high risk of developing OHSS [1–3]. In contrast to human chorionic gonadotropin (hCG) triggering, the GnRH agonist induces a surge mimicking the natural surge of gonadotropins, with the consequent short-lasting elevation of both Luteinizing hormone (LH) and follicle-stimulating hormone (FSH). While the precise mechanism of OHSS prevention is not thoroughly understood, the leading hypotheses is that the shorter duration of the endogenous LH surge induced by GnRH agonist, compared with the sustained high levels of hCG, results in shorter and milder secretion of vasoactive substances, such as vascular endothelial growth factor (VEGF) which, along with other proinflammatory cytokines, known to play a fundamental role in the pathophysiology of OHSS [4, 5]. Although previous studies described similar embryologic outcomes when comparing the hCG and GnRH agonist for ovulation triggering [6, 7], a poorer clinical outcome was observed with fresh embryo transfer (lower clinical pregnancy rate and higher early pregnancy loss rate) after the GnRH-agonist trigger [6, 8, 9]. These results were attributed to early lysis of the corpus luteum and subsequent luteal phase insufficiency with reduced progesterone levels. The type and mode of triggering final follicular maturation have different effect of IVF outcome [7]. Recently, Haas et al. described improved IVF outcomes using the dual trigger (hCG + GnRH agonist) for final follicular maturation, including an increased number of oocytes including mature oocytes and an increased number of blastocysts (total and top-quality) compared to triggering with hCG alone [10]. The apparently beneficial effect of the dual trigger might be explained by the more physiologic surge of FSH with the addition of GnRH agonist to the hCG trigger, and a potential beneficial effect on the mural and cumulus granulosa cells (GCs) surrounding and supporting the oocyte. The combined trigger may potentially lead to alterations in gap junctions, FSH and LH receptors, and epidermal-like growth factor in GCs [11, 12]. On the other hand, the GnRH agonist alone or added to hCG in the dual trigger might cause increased apoptosis of GC as previously described in vitro [13]. In the present study, our objective was to evaluate the in vivo effect of different modes of triggering on the degree of apoptosis of GCs and the potential effect on progesterone secretion.

The research was approved by the research ethics review board at Mount Sinai Hospital, Sinai Health System, and written informed consent was obtained from each participating subject. The 30 patients included in the study ( 10 in each arm) were women < 43 years of age undergoing IVF due to male factor infertility, tubal factor, or for the purpose of preimplantation genetic diagnosis (PGD, genetic tests of embryos prior implantation). Patients with infertility related to female pathology, such as endometriosis or poor ovarian response were excluded from the study. Intracytoplasmic sperm injection (ICSI) was performed on all the patients due to a clinic policy. The decisions regarding treatment protocol including the mode of triggering were made according to physician preference. The follicular fluid (FF) was obtained at the time of oocyte retrieval for IVF procedures. The women were treated using the antagonist protocol. Ovarian stimulation with a daily SC dose of recombinant FSH (Gonal F, EMD Serono) was started on the third day of the menstrual cycle, and after 5 days of stimulation, it was replaced with highly purified human menopausal gonadotropin (hMG) (Menopur, Ferring). The amount of the initial dose depended on the age, body mass index (BMI), basal antral follicle count, hormone level, and treatment history. When three leading follicles reached 17 mm or above in diameter, the women received either GnRH agonist alone (Suprefact 0.5 mg), hCG alone (Pregnyl 10,000IU), or a dual trigger (GnRH agonist+ hCG) for final oocyte maturation. Oocyte retrieval was performed by transvaginal ultrasound-guided needle aspiration 36 h post trigger. Isolation of granulosa-luteal cells The mural granulosa-luteal cells were isolated from follicular fluid and purified according to previously described methods [14–16]. Briefly, the follicular aspirate was centrifuged at 700g for 10 min at 4 °C, The top cellular layer was collected and washed in phosphate-buffered saline (PBS without Ca or Mg) up to a total volume of 5 ml, then layered on top of 5 ml of a Ficoll-Paque 100% Ficoll gradient cushion (GE Healthcare, Mississauga, ON, Canada) and centrifuged at 700×g for 20 min at 4 °C. GCs at the interface of the Ficoll and the buffer were aspirated, re-suspended in 15 ml of 1× PBS, and centrifuged at 700×g for 10 min at 4 °C. The cell pellet was collected and fixed with 500–1000 μl of formalin (depending on the pellet size), suspended and kept at 4 °C for analysis. Before use, cell suspension was smeared on a pre-charged slide, let briefly air dry, and used either for TUNEL or IHC. TUNEL DNA fragmentation in the granulosa-luteal cells was measured using the method of terminal deoxynucleotidyl transferase (TdT)–mediated dUPT biotin end labeling (TUNEL) as previously described [17]. The percentage of GC undergoing apoptosis was determined by dividing TUNEL positive GC by total GC as determined by 4′6-diamidino-2-phenylindole (DAPI) staining. Immunohistochemistry Granulosa-luteal cells were treated for immunohistochemistry as previously described [17]. Anti-active-caspase-3 antibody (1:400; Cell Signaling, cat# 9661) was diluted in 4% horse serum and incubated overnight at 4 °C. After washing, secondary antibody donkey anti-rabbit Alexa 594 (Invitrogen, 1:1000) was applied for 1h at room temperature. Upon washing, final nuclear counterstain was performed with DAPI and cells were counted on Zeiss Axioplan Microscope using appropriate light filters. We used negative control (GCS without anti-active-caspase-3 antibody, incubated with the secondary antibodies) for validation. Granulosa-luteal cell culture and progesterone measurement The granulosa-luteal cells were collected and washed with PBS as described above, counted and plated in 24-well plates at a density of 100,000 cells/well, suspended in basic medium (medium 199; Sigma-Aldrich) supplemented with 5% FCS (Invitrogen), 1% penicillin/streptomycin (Gibco), and incubated at 37 °C in a humidified atmosphere with 5% CO2 in air. The cells were cultured for 24 h, and the medium was collected. Progesterone concentrations were measured via ELISA kit (Enzo) according to the manufacturer’s instructions. Statistical analysis Normality of the data distribution was tested using the Shapiro-Wilk or Kolmogorov-Smirnov tests. Continuous variables were presented as mean ± standard deviation (STD). Comparison between the variables was conducted with ANOVA test or Kruskal-Wallis test. We performed a post hoc test (Tukey HSD) comparing the apoptosis levels between the three groups. The significance threshold was set as p < 0.05. Statistical analyses were conducted using the IBM Statistical Package for the Social Sciences (IBM SPSS Statistics for windows V22.0; IBM Corporation Inc., Armonk, NY, USA).

Thirty patients were included in the study, ten in each trigger arm. There were no differences between the three groups (hCG vs. GnRH agonist vs. dual trigger) in terms of etiology for fertility treatments, basal FSH, BMI, the age, the length and doses of stimulation, and the number of oocytes retrieved (Table 1). We performed ANOVA test and found that the proportion of apoptotic cells was significantly higher in the GnRH agonist–alone group compared to hCG-alone and the dual trigger groups (13.5 ± 1.5% vs. 7.8% ± 1.8 vs. 10.1% ± 2, respectively, P < 0.01) (Fig. 1). Moreover, the expression of active-caspase-3 was also significantly increased in the GnRH agonist–alone group compared with the hCG-alone and the dual trigger groups (15.5% ± 2.9 vs. 8.4% ± 1.6 vs. 12.7% ± 2.6, respectively P < 0.01) (Fig. 2). Table 1. | GnRH agonist trigger | Dual trigger | hCG trigger | p | | |---|---|---|---|---| | Number of patients | 10 | 10 | 10 | | | Age | 34.7 ± 4.6 | 36.1 ± 3.7 | 33 ± 4 | 0.4 | | BMI | 23.7 ± 2.2 | 25.7 ± 6.8 | 26.9 ± 8 | 0.7 | | Basal FSH (IU) | 7.1 ± 1.6 | 8.8 ± 3.7 | 7.1 ± 2.4 | 0.5 | | Days of stimulation (n) | 10.5 ± 1/5 | 9.7 ± 1.4 | 9.7 ± 0.7 | 0.8 | | Total dose of gonadotropins (IU) | 2875 ± 907 | 3121 ± 1379 | 2681 ± 1187 | 0.3 | | Oocytes (n) | 12.5 ± 3.5 | 11.2 ± 5.3 | 10.3 ± 4.7 | 0.7 | We then performed a post hoc test (Tukey HSD) comparing the apoptosis levels between the three groups. TUNEL: We compared the GnRH agonist group with the dual trigger group 13.5 ± 1.5% vs. 10.1% ± 2, p = 0.001), the GnRH agonist with the hCG group (13.5 ± 1.5% vs. 7.8% ± 1.8, p < 0.001) and the dual trigger groups with the hCG group (10.1% ± 2 vs. 7.8% ± 1.8, p = 0.02). Immunohistochemistry: We compared the GnRH agonist group with the dual trigger group (15.5% ± 2.9 vs. 12.7% ± 2.6, p = 0.04), the GnRH agonist with the hCG group (15.5% ± 2.9 vs. 8.4% ± 1.6, p < 0.001), and the dual trigger groups with the hCG group (12.7% ± 2.6 vs. 8.4% ± 1.6 p = 0.002). The progesterone levels measured in the granulosa-luteal cell culture medium after 24 h of incubation were similar between the three groups (3422 pmol/mil ± 913 vs. 3181 pmol/mil ± 582 vs. 3515 pmol/mil vs. ± 762 p = 0.8).

Although it is known that the GnRH agonist trigger leads to luteolysis with consequent corpus luteum insufficiency, the precise mechanism is not well-understood. It has been proposed that the short duration of the endogenous LH surge induced by the GnRH agonist trigger, followed by a withdrawal of the LH support to the corpus luteum might lead to an early corpus luteum demise [18]. It is also well-known that GnRH agonist triggering of final follicular maturation is successful with comparable numbers of MII oocytes, comparable fertilization rates, and top-quality embryo development, as compared to cycles triggered with hCG. These findings imply that the LH and FSH surge induced by GnRH agonist is sufficient to induce luteinization of the follicle with transition of steroidogenesis from predominantly estrogen to estrogen and progesterone. Thus, it is not surprising that the granulosa-luteal cells aspirated at the time of oocyte retrieval produce progesterone to the same extent in all three groups in the present study. It is later on in the luteal phase that steroidogenesis declines with GnRH agonist trigger, possibly because of the short duration of the LH surge, but as seen in the present study, because of apoptosis of granulosa-luteal cells and subsequent premature demise of the corpus luteum. The observation of elevated proportion of apoptotic granulosa-luteal cells as early as 36 h after the GnRH agonist trigger, when there should still be LH support present, suggests a possible direct effect of the agonist on these cells despite in vitro evidence to the contrary with regard to steroidogenesis [19, 20]. Previous studies have shown that GnRH agonist in vitro directly affect apoptosis of ovarian cells (involving caspase 3, caspase 8, and caspase 9), but its mechanism is not clearly understood [13, 21–23]. To mention, the aforementioned studies involved granulosa cells cultured with GnRH agonist and not granulosa cells retrieved from patients triggered with GnRH agonist—as performed in our current study. Engmann et al. also aimed to evaluate in vivo the viability of granulosa-luteal cells obtained from patients triggered with GnRH agonist vs. hCG and assessed the secretion of steroids by cultured granulosa cells. In contrast to our findings, they did not observe any difference in the proportion of granulosa cell apoptosis between the two groups, and similar to our findings, the progesterone levels in the medium of the cultured granulosa cells were similar [24]. Engmann et al. detected apoptosis with different methods. Identification of apoptotic nuclei was achieved by in situ DNA staining done by adding hydroethidine directly to the culture medium. Under these conditions, only cells with condensed or fragmented nuclei were intensely stained with hydroethidine. In our research, we detected apoptosis via TUNEL and immunohistochemistry. The TUNEL assay method relies on the enzyme terminal deoxynucleotidyl transferase (TdT), which attaches deoxynucleotides to the 3′-hydroxyl terminus of DNA breaks. The immunohistochemistry relies on anti-active-caspase-3 antibody detecting the apoptotic cells We believe that the different methods of detecting apoptosis may be the explanation for the different results.

of the study First, it was not randomized and might be subject to selection bias. Second is small sample size. Third, further apoptosis may occur after more than 36 h following trigger. The progesterone levels in the culture media were measured after 24 h and not after a longer time which could have an impact on the results. The TUNEL assay shows relatively low specificity for detecting apoptosis which might affect the results. In the present study, we observed significant increased levels of apoptosis in GCS retrieved following GnRH agonist trigger/dual trigger. The increased apoptosis did not correlate with a similar decrease in progesterone levels secreted by the GCS in culture. It might be therefore speculated that GnRH agonist induced apoptosis is one of the explanation for the subsequent premature corpus luteum demise post GnRH agonist trigger. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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