Evaluating the Impact of Long-Term GnRH Agonist Therapy on Pregnancy Outcomes in Endometriosis-Associated Implantation Failure and Pregnancy Loss

Endometriosis, recurrent implantation failure, recurrent pregnancy loss, GnRH agonist, embryo transfer https://doi.org/10.46989/001c.115593 Journal of IVF-Worldwide Vol. 2, Issue 1, 2024 Purpose This study aimed to investigate the efficacy of long-term gonadotropin-releasing hormone (GnRH) agonist therapy in preventing endometriosis progression and relieving symptoms, particularly on pregnancy outcomes during thawed embryo transfer in patients experiencing endometriosis and recurrent implantation failure or recurrent pregnancy loss.

In individuals with clinical endometriosis and a history of recurrent implantation failure or recurrent pregnancy loss, we conducted a comparative analysis of clinical outcomes between those undergoing long-term GnRH agonist treatment for symptom relief, such as menstrual pain, followed by embryo transfer using Hormone Replacement Therapy (HRT) cycle, and those undergoing embryo transfer using an HRT cycle without GnRH agonist treatment. The study examined various clinical outcomes between the two groups.

The primary outcomes included live birth rate (LBR), miscarriage rate, biochemical pregnancy rate, and perinatal complications. The GnRH agonist group showed significantly higher LBR than the control group (37.50% vs. 13.04%; p=0.02). Multivariable logistic regression analysis, adjusted for age and gravidity, showed significantly higher LBR in the GnRH agonist group compared to the control group (odds ratio: 15.3; 95% confidence interval: 2.30, 102.00; p=0.005).

The findings of this study suggested that employing a GnRH agonist in the embryo transfer protocol is effective for patients with endometriosis experiencing recurrent implantation failure or recurrent pregnancy loss.

Endometriosis, affecting approximately 10% of women in their reproductive years,1 is a condition characterized by pain, notably menstrual cramps and infertility. Nearly half of patients with endometriosis experience infertility, with the disease diagnosed in 20-25% of individuals facing fer- tility challenges. The fertility rate in women with en- dometriosis decreases from 0.15-0.2 women/month to 0.02-0.10 women/month.2 Additionally, the likelihood of endometriosis is six to eight times higher in infertile women compared to their fer- tile counterparts.3 Reported causes of infertility in women with endometriosis encompass luteinized unruptured folli- cle syndrome, impaired oocyte and embryo formation, en- docrine disorders involving luteinizing hormone (LH) surge abnormalities, and ovulation disorders.4 Moreover, inflam- mation in the pelvic region is identified as a potential con- tributor to these conditions. Corresponding author, Email: [email protected] a Kobanawa M. Evaluating the Impact of Long-Term GnRH Agonist Therapy on Pregnancy Outcomes in Endometriosis-Associated Implantation Failure and Pregnancy Loss. Journal of IVF-Worldwide. 2024;2(1):29-36. doi:10.46989/001c.115593 Studies have reported elevated levels of inflammatory cytokines in the pelvic region of individuals with en- dometriosis.5,6 Moreover, studies indicate an increased number of macrophages and cytokines in the peritoneal fluid of women with endometriosis, with this fluid demon- strated to inhibit sperm and oviduct cilia function in vitro.7 In individuals with endometriosis, elevated concentra- tions of IL-6 are observed in intraperitoneal reservoir fluid, blood, and follicular fluid. Studies have reported that high IL-6 levels in intraperitoneal fluid can suppress early em- bryonic development in mice, reduce human sperm motil- ity, and inhibit estrogen production by granulosa cells. These findings imply the involvement of increased cy- tokines in ascites and blood from individuals with en- dometriosis in reducing fertility.8 Furthermore, the reduced expression of decidualization markers in endometrial tissue from patients with en- dometriosis, compared to endometriosis-free en- dometrium, suggests the potential influence of cytokines, increased in ascites fluid, on these markers.9 These suggest a potential association between endometriosis and implan- tation failure. Increased miscarriage rates have also been reported among patients with endometriosis.10,11 Moreover, it has been indicated that a higher miscarriage rate exists among patients with endometriosis compared to those without en- dometriosis, particularly in cases involving assisted repro- ductive technology (ART).12 These findings suggest that patients with endometriosis may exhibit abnormal en- dometrial conditions due to inflammatory cytokines and other factors, leading to recurrent implantation failure and repeated miscarriages. Furthermore, several studies have reported that GnRH agonist ameliorates pelvic inflamma- tion in endometriosis.13,14 In this study, we retrospectively examined whether the use of GnRH agonists in HRT cycles, with or without em- bryo transfer, influences the clinical outcomes for patients undergoing ART due to recurrent implantation failure (RIF) (defined as failure to conceive after three or more successful embryo transfers)15 or recurrent pregnancy loss (RPL) (de- fined as two or more miscarriages).16 The primary focus of the retrospective examination was to assess if the use of GnRH agonists during embryo transfers in HRT cycles could bring about changes in the clinical outcome.

Seventy patients, all with a history of endometriosis and diagnosed with recurrent implantation failure or recurrent pregnancy loss, were included in this study based on their previous medical history. The clinical outcomes of eligible patients who underwent thawed embryo transfer of frozen blastocysts by HRT cycle in assisted reproductive medicine between April 2022 and December 2022 were retrospec- tively analyzed. The diagnosis of endometriosis was based on subjective symptoms such as dysmenorrhea, deep dys- pareunia, and dysuria, with Beecham Classification stage II or higher considered indicative of clinical endometriosis, relying on internal examination findings and transvaginal ultrasound.17 For the purposes of this study, RIF was defined as the inability to achieve pregnancy after three or more embryo transfers involving good blastocysts (Gardner’s classifica- tion 3BB or higher). RPL was categorized for patients who had encountered two or more previous miscarriages. Ethi- cal approval was obtained from the Ethical Review Commit- tee, and the study was performed on cases where informed consent was obtained from the patients. For controlled ovarian stimulation, beginning on the first three days of menstruation, either Follitropin alfa (Go- nal F; Merck Biopharma, Tokyo, Japan) or Follitropin delta (REKOVELLE; Ferring Pharma, Tokyo, Japan) was used in performing the antagonist or progestin-primed ovarian stimulation (PPOS) method. Upon reaching approximately 14 mm, the primary folli- cle triggers the initiation of a GnRH antagonist (Ganirest; MSD, Tokyo, Japan) at a dose of 0.25 m/day, which was maintained until the primary follicle attained a size of 18-20 mm. The gonadotropin and GnRH antagonist 0.25 m/day were discontinued upon reaching this size. Simul- taneously, on the same day, 250 μg of recombinant hCG (Ovidrel; Merck biopharma) or 600 μg of a GnRH agonist (Suprecur; CLINIGEN, Tokyo, Japan) was administered to trigger oocyte maturation. Oocytes retrieval was performed 34 to 36 hours after the triggering. Oocytes were insemi- nated with 1 to 5 pre-cultured oocytes at a sperm concen- tration of 10 × 104/ml. The following morning (approxi- mately 19 hours post-insemination), oocytes were denuded using an inverted microscope (OLYMPUS IX73, Tokyo, Japan), and a clean bench in the intracytoplasmic sperm injection (ICSI). Sperm collected after swim-up were used in 10% polyvinylpyrrolidone (Irvine Scientific, Santa Ana, U.S.A.) to achieve the optimal concentration. The retrieved oocytes underwent pre-culture and denudation using Cu- mulus Remover (KITAZATO, Shizuoka, Japan). After ICSI, fertilization was confirmed the next morning (approxi- mately 18 hours post-ICSI). Embryos were cultured individ- ually in ART CULTURE DISH 12 or 24 (nipro, Tokyo, Japan), with 50 μL CSCM-NX per well. Blastocysts were cultured until Day 5/Day 6 and classified as good blastocysts accord- ing to Gardner’s classification 3BB or higher before under- going freezing. Vitrification media (KITAZATO) served as the embryo freezing medium, and CRYO-TOP (KITAZATO) as the device. The GnRH agonist group received depot leuprolide ac- etate (Leuprorelin Acetate; ASKA Pharmacy, Tokyo, Japan) for 1 to 2 months from the 1st to 3rd day of any menstrual period to relieve symptoms such as menstrual cramps as- sociated with endometriosis, then administration of the estrogen preparation (ESTRANA Tapes; Hisamitsu Phar- maceutical, Saga, Japan) 0.72 mg x 4/every other day was started without waiting for the onset of menstruation. Upon confirming endometrial thickness exceeding 7 mm, progesterone drugs (LUTINUS Vaginal Tablets: Ferring Pharma) at 100 mg thrice a day was initiated (P＋0). At six days initiating progesterone drug(P+5), after thawing, the embryos were cultured for 4-6 hours and then trans- Evaluating the Impact of Long-Term GnRH Agonist Therapy on Pregnancy Outcomes in Endometriosis-Ass… Journal of IVF-Worldwide 2 Table 1. Patient Characteristics Characteristics GnRH agonist group (n=24) Control group (n=46) p-value Age at oocytes pick up, age 38.04±5.61 35.22±4.38 0.02 Age at embryo transfer, age 38.71±5.92 37.39±4.76 0.31 boy weight, kg 58.05 [44.00, 75.00］ 57.00 [41.80, 95.00］ 0.90 BMI, kg/m2 23［18.00, 33.00］ 22.5［17.00, 34.00］ 0.50 AMH, ng/ml 2.57［0.20, 10.99］ 3.29［0.02, 17.64］ 0.07 Gravidity, times 1.00［0.00, 3.00］ 1.00［0.00, 4.00］ 0.39 Parity, times 0.00［0.00, 1.00］ 0.00［0.00, 2.00］ 0.009 Number of past embryo transfers, times 4.00 [2.00, 29.00] 4.00 [2.00, 16.00] 0.24 Duration of estrogen supplementation until embryo transfer, days 19.00±2.04 17.93±1.73 0.02 Thickness of the endometrium, mm 9.88±1.36 10.27±1.52 0.29 Serum estradiol level on day of progesterone start, pg/ml 275.12±178.64 299.35±158.23 0.56 Serum progesterone levels on the day of embryo transfer, ng/ ml 10.89±2.71 11.64±5.09 0.50 In vitro fertilization (%) 16 (66.70) 20 (43.50) 0.08 Intracytoplasmic sperm injection (%) 8 (33.30) 26 (56.50) 0.08 Blastocyst Quality Score, points 28.83±10.08 31.11±8.53 0.32 Data are presented as mean ± standard deviation or median [Max, min] or n, n (%). The t-test or Mann-Whitney’s U test was used for patient characteristics. ferred under transvaginal ultrasound guidance. In the con- trol group, embryos were transferred without depot leupro- lide acetate in HRT cycles initiated on days 1-3 of any menstrual period. In order to evaluate the effects of GnRH agonist, only patients who underwent embryo transfer during HRT cycles were included in the control group, regardless of menstrual cycle abnormalities or ovulation disorders. Clinical out- comes, including live birth rate, miscarriage rate, biochem- ical pregnancy rate and perinatal complications, were com- pared following thawed embryo transfer. In this study, biochemical pregnancies were defined with a cut-off as in- creasing values of β-hCG > 5 IU/L not confirming fetal sac by transvaginal ultrasonography.18 Statistical analysis employed a t-test or Mann-Whitney’s U test for patient characteristics, with a significance set at a p < 0.05. The χ2 test was used for clinical outcomes, with a significance set at p < 0.05. Additionally, a logistic regression model, adjusted for confounding factors, was used to evaluate the effect of em- bryo transfer with GnRH agonist on the live birth rate (LBR). All statistical analyses were performed using EZR (Saitama Medical Center, Saitama, Japan), a software that extends R’s capabilities (The R Foundation for Statistical Computing, Vienna, Austria).

A total of 70 patients (GnRH agonist group, n=24; control group, n=46) diagnosed with endometriosis-associated with RIF or RPL were included in the analysis. The demographic characteristics of the women are presented in Table 1. To facilitate the comparison, the grades of transferred em- bryos were converted to continuous variables using the Blastocyst Quality Score.19 Patient characteristics were generally similar between the groups. However, differences were observed in age at oocyte pick-up (p = 0.02), parity (p = 0.009), and duration of estrogen supplementation until embryo transfer (p = 0.02) (Table 1 ). The clinical results indicated that LBR was significantly higher in the GnRH agonist group compared to the control group (p = 0.02), and the biochemical pregnancy rate was significantly lower in the GnRH agonist group (Table 2 ). The univariate logistic analysis showed a significant as- sociation between LBR and age at oocyte pick-up and gra- vidity (Table 3 ). In the multiple logistic regression analysis, adjusted for age at oocytes pick up and gravidity, the LBR was signifi- cantly higher in the GnRH agonist group than in the con- trol group (odds ratio: 15.3; 95% confidence interval: 2.30, 102.00; p = 0.005) (Table 4 ).

This study suggests that utilizing a GnRH agonist in embryo transfer with HRT cycle may be effective for patients with endometriosis experiencing RIF or RPL. Similar findings have been reported in several previous studies. Studies have indicated that the long-term use of GnRH agonists prior to in vitro fertilization and embryo transfer (IVF-ET) in patients with endometriosis results in signifi- cantly higher rates of ongoing pregnancy compared to stan- dard controlled ovarian stimulation regimens.20 Similarly, the long-term use of GnRH agonists before IVF/ICSI in in- Evaluating the Impact of Long-Term GnRH Agonist Therapy on Pregnancy Outcomes in Endometriosis-Ass… Journal of IVF-Worldwide 3 Table 2. Comparison of clinical outcomes. GnRH agonist group (n=24) Control group (n=46) p-value Live birth rate (%) 9 (37.50) 6 (13.04) 0.02 Miscarriage rate (%) 0 (0.00) 1 (14.20) 0.47 Biochemical pregnancy rate (%) 0 (0.00) 10 (21.74) 0.01 Perinatal complications (%) 1 (4.20) 0 (0.00) 0.34 The data are presented as n, n (%). The chi-squared test was used to analyze the group difference. Table 3. Univariate logistic regression analysis for live birth rate. Variables Adjusted OR(95%CI) p-value Age at oocytes pick up 0.84 (0.74, 0.96) 0.01 Age at embryo transfer 1.02 (0.91, 1.15) 0.68 boy weight 1.03 (0.98, 1.09) 0.26 BMI 1.13 (0.99, 1.29) 0.06 AMH 0.98 (0.79, 1.21) 0.82 Gravidity 0.30(0.12, 0.77) 0.01 Parity 0.25 (0.05, 1.21) 0.09 Number of past embryo transfers 1.07 (0.95, 1.20) 0.27 Duration of estrogen supplementation until embryo transfer 1.17 (0.85, 1.60) 0.32 Thickness of the endometrium 1.17 (0.81, 1.70) 0.41 Serum estradiol level on day of progesterone start 1.00 (0.99, 1.00) 0.70 Serum progesterone levels on the day of embryo transfer 0.97 (0.85, 1.11) 0.67 In vitro fertilization 1.27 (0.41, 4.00) 0.68 Intracytoplasmic sperm injection 1.27 (0.41, 4.00) 0.68 Blastocyst Quality Score 0.98 (0.92, 1.05) 0.61 CI, confidence interval OR, Odds ratio Table 4. Multivariable logistic regression analysis results of the two groups and clinical outcomes. Variables OR(95%CI) p-value GnRH agonist group vs Control group 15.3 (2.3, 102.00) 0.005 Age at oocytes pick up 0.72 (0.58, 0.88) 0.002 Gravidity 0.18 (0.04, 0.70) 0.01 fertile women with endometriosis or adenomyosis has been associated with increased ongoing pregnancy rates in both fresh and frozen cycles.21,22 Possible causes of implantation failure and miscarriage in patients with endometriosis include progesterone (P4) resistance, abnormal expression of integrin (a cell adhesion factor), and overexpression of B-cell lymphoma 6 (BCL6), a biomarker associated with endometriosis.23 Estrogen re- ceptors (ER-α) in the endometrium, responding to estrogen during the proliferative phase and decreasing during the se- cretory phase in response to P4, play a crucial role in im- plantation.24 Suppression of ER-α is important for implan- tation,25 and P4 is known to promote downregulation of estrogen receptors in the endometrium and promote decid- ualization.26‑28 In endometriosis, abnormal expression of aromatase leads to a local increase in estrogen in the en- dometrium,29 causing resistance to P4.30 Such abnormal- ities in estrogen metabolism may affect endometrial re- ceptivity, leading to downregulation of P4 receptors and upregulation of estrogen receptors, resulting in decidual- ization.31 The absence of progesterone receptor B (PR-B), one of the P4 receptor isoforms in endometriosis, has been suggested as a potential cause of these abnormalities in es- trogen metabolism.32 Progesterone receptor (PGR) has isoforms PR-A and PR- B, with PR-A acting as a major repressor of PR-B, whereas PR-B tends to be a more potent activator of P4 target genes.33,34 P4 resistance in endometriosis tissue may be ex- plained by the presence of the inhibitory PR isoform PR-A and the absence of the active isoform PR-B.35,36 GnRH ag- Evaluating the Impact of Long-Term GnRH Agonist Therapy on Pregnancy Outcomes in Endometriosis-Ass… Journal of IVF-Worldwide 4 onists inhibit aromatase activity in the endometrium, pre- venting local estrogen increase.37 This effect helps prevents upregulation of estrogen receptors and the active P4 re- ceptor, PR-B, potentially improving decidualization and ad- dressing implantation failure. Another biomarker indicative of the embryonic receptive state during the implantation phase of the endometrium is the cell adhesion factor integrin, particularly ανβ3 inte- grin. Expression of ανβ3 integrin has been linked to im- plantation failure.38‑42 Moreover, endometriosis has been associated with significantly higher levels of ER-α expres- sion in the mid-secretory endometrium, leading to reduced αvβ3 integrin expression.25 The administration of long-acting GnRH agonists prior to treatment has been shown to enhance αvβ3 integrin ex- pression in patients with endometriosis, potentially con- tributing to improved implantation and pregnancy contin- uation rates.22,43 Patients with endometriosis often exhibit a high levels of BCL6 protein, with a positive predictive value of 96%. BCL6 is a protein encoded by a proto-oncogene located on chromosome 3 (3q27.3) that stimulates inflammatory cy- tokines, such as IL-6. Inflammation can lead to P4 resis- tance and abnormal decidualization.44 Similarly, overex- pression of BCL6 protein is associated with P4 resistance by interfering with P4 signaling, a crucial factor for decidu- alization.45 Studies have reported a significantly lower live birth rate in ART for patients with elevated BCL6 (11.5%) compared to those without elevated BCL6 (58%).46 Women suspected of having endometriosis with abnor- mal endometrial BCL6 expression have demonstrated sig- nificantly improved fertility following two months of GnRH agonist or surgical treatment.47 These findings suggest that in patients with endometriosis, RIF and RPL may result from P4 resistance due to abnormal estrogen metabolism, decreased integrin expression, and excessive expression of BCL6 protein. The use of GnRH agonist as a premedication for 1-2 months may improve the clinical outcomes for these patients. Considering that the ESHRE RPL guidelines encompass biochemical pregnancies in the definition of RPL,16 all pa- tients with implantation failure in this study had experi- enced at least two biochemical pregnancies, this study sug- gests that embryo transfer during HRT cycles with GnRH agonist can improve LBR and decrease biochemical preg- nancy rate for RPL patients in a broad sense.

The study was conducted with a limited number of patients with uterine infertility, and a notable limitation is that the diagnosis of endometriosis included patients clinically di- agnosed according to the Beecham classification,17 which encompasses a group where a definitive histological diag- nosis of endometriosis has not been established. However, in recent years, it is not always necessary to perform la- paroscopy and histological examination, as a therapeutic intervention is recommended when clinically suspicious findings of endometriosis are identified during an internal examination or transvaginal ultrasound.48‑50 Given the ret- rospective nature of this study, future prospective studies, with or without prior administration of GnRH agonists in patients with endometriosis, are warranted. ACKNOWLEDGMENTS We want to thank Editage (www.editage.com) for editing the English language. STATEMENTS AND DECLARATIONS} The authors have no conflicts of interest to declare. All procedures were conducted in accordance with the ethical standards of the responsible committee on human experi- mentation (facility and national levels) and adhered to the Helsinki Declaration of 1964 and its subsequent amend- ments. Informed consent was obtained from all patients in- cluded in the study. This article does not involve any stud- ies conducted by authors on animal subjects. The protocol for the research project, which includes human subjects, has been approved by the Medical Corporation Kobanawa Clinic Ethic Screening Committee. COMPETING INTERESTS The authors have no competing interests to disclose. Submitted: January 15, 2024 CDT, Accepted: March 22, 2024 CDT This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CCBY-NC-SA-4.0). View this license’s legal deed at https://creativecommons.org/licenses/by-nc-sa/4.0 and legal code at https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode for more information. Evaluating the Impact of Long-Term GnRH Agonist Therapy on Pregnancy Outcomes in Endometriosis-Ass… Journal of IVF-Worldwide 5

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