Differences of pro-inflammatory and cell apoptosis markers between patients with normal and poor ovarian response during in vitro fertilization program Running Titel: Pro-inflammatory markers in IVF patients

doi:10.21203/rs.3.rs-4894275/v1

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Differences of pro-inflammatory and cell apoptosis markers between patients with normal and poor ovarian response during in vitro fertilization program Running Titel: Pro-inflammatory markers in IVF patients

https://doi.org/10.21203/rs.3.rs-4894275/v1

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This study aimed to compare pro-inflammatory (Interleukin-6; IL-6 and TNF-α) and cell apoptosis (GABRA-1) markers in patients with normal ovarian response (NOR) and poor ovarian response (POR) to ovarian stimulation during in vitro fertilization (IVF) treatment. A cross-sectional study was conducted with 27 patients (NOR = 16 and POR = 11). Pleiotropic cytokine IL-6 levels in follicular fluid were determined by enzyme-linked immunosorbent assay. Relative expression levels of TNF-α and GABRA-1 related genes were determined in cumulus cells by reverse transcription-quantitative polymerase chain reaction. TNF-α levels were significantly higher in POR group compared to NOR group (POR = 7.1 ± 1.9 and NOR = 2.4 ± 1.5, P < 0.001). Additionally, positive correlation was noted between TNF-α relative expression levels and factors associated with ovarian response, including levels of anti-müllerian hormone (AMH) (r² = 0.69, P = 0.008), antral follicle count (r² = 0.73, P < 0.001) and number of retrieved oocytes (r² = 0.74, P < 0.001). However, IL-6 and GABRA-1 levels did not differ between cohorts. In conclusion, TNF-α presents significantly different between NOR and POR patients, suggesting its crucial role in ovarian response during IVF treatment. This finding provides an opportunity for further study of POR pathogenesis.

Health sciences/Biomarkers

Health sciences/Diseases

Health sciences/Medical research

In vitro fertilization

Normal ovarian response

Pro-inflammatory marker

Poor ovarian response

Poor ovarian response (POR) to ovarian stimulation during in vitro fertilization (IVF) treatment has remained one of crucial factors leading to diminished ovarian reserve and cycle cancellation¹. According to the previous published data, the incidence of POR in women undergoing the ovarian stimulation programs ranges from 5.6–35.1%². Although the cause of POR pathology might be attributed to advanced maternal age³, the other aspects such as genetic background, environmental or iatrogenic factors could present the adverse effects to the ovarian response during the ovarian stimulation⁴. Recently, the other regulatory factors related to POR incidence, including oxidative stress and pro-inflammatory cytokines were focused, primarily in micro-environment surrounding oocytes^{5, 6}. The cytokines are a group of protein cell regulators that fall into three categories: lymphokines, chemokines, and interleukins⁷. They play pivotal roles in several physiological processes, such as immune regulatory and inflammatory response which are involved in wide range of diseases⁷.

Regarding the inflammatory response complex, Interleukin-6 (IL-6) has been considered multifunctional cytokines which could trigger cell function via membrane receptors (i.e. sIL-6R alpha) resulting in intracellular activation of JAK/STAT cascade⁸. According to human ovarian physiology, IL-6 protein expression and IL-6 level have been observed in granulosa cells⁹ and follicular fluid¹⁰, respectively. Elevation of IL-6 level in the follicular fluid was related to low number of oocyte retrieval in women undergoing IVF⁵ and poor oocyte development competency in mouse study¹¹. To date, the scientific data has demonstrated that IL-6 could be altered by either genetics or epigenetics. For example, the previous study in the patients with endometriosis revealed that IL-6 synthesis in endometriotic cells could be triggered by TNF-α mRNA function via NF-kappa B and MAPK pathways¹². TNF-α is a pro-inflammatory cytokine known for its critical roles in cell signaling pathways that regulate inflammation, cellular proliferation, and apoptosis¹³. According to ovarian physiology, TNF-α has been implicated in the regulation of follicular atresia¹⁴ and luteolysis¹⁵, processes essential for maintenance of follicular health and competence. In addition, IL-6 expression could be regulated by the epigenetics (i.e. non-coding RNA; miRNA-139¹⁴) while miRNA-139 could regulate IL-6 function leading to decreasing of cell survival rate via its target gene (GABRA-1)¹⁶.

To date, the data in consequence of candidate genes that might relate to IL-6 regarding the ovarian stimulation response has not been elucidated. Therefore, the present study principally aimed to compare the levels of pro-inflammatory (IL-6 and TNF-α) and cell apoptosis (GABRA-1) biomarkers in biological samples (follicular fluid and cumulus cells) retrieved from the patients with normal ovarian response (NOR) and POR.

Ethical approval

This cross-sectional study was approved by Institutional Review Board, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand on January 18, 2022 (IRB No. 905/64). The written informed consents were obtained from all participants. Additionally, the authors confirmed that this experiment was performed in accordance with relevant guidelines and regulations.

Participant population

Twenty-seven of women who underwent ovarian stimulation and IVF programs at Reproductive Biology Unit, Department of Obstetrics and Gynecology, Faculty of Medicine Chulalongkorn University and King Chulalongkorn Memorial Hospital from March 2022 to December 2022 were included. The patients were classified as normal ovarian response (NOR; n = 16 with age range 31-40 years old) and poor ovarian response (POR; n= 11 with age range 33-42 years old) to the ovarian stimulation program. The poor ovarian response patients included in this study must present at least 2 of 3 features, including advanced age (≥ 40 years old) or 1-4 retrieved oocytes on oocyte pick up (OPU) day or abnormal ovarian reserve test (i.e. number of antral follicle counts (AFC) < 5-7 follicles or < 0.5-1.1 ng/mL of anti-müllerian hormone (AMH). The patients with ovarian pathology such as polycystic ovary syndrome or basal follicle stimulating hormone (FSH) level over 20 IU/L or women with any acute or chronic inflammatory and autoimmune diseases were excluded.

Ovarian stimulation protocol

The ovarian stimulation protocol was managed regarding each individual baseline characteristics under judgement of physicians. The patients from both groups were treated with gonadotrophin-releasing hormone (GnRH) antagonist protocol. Initial dose of 200-300 U/day recombinant follicle stimulating hormone (rFSH) (Follitropin alpha; Gonal-f^®, Merck Serono SA, Switzerland) or Follitropin Beta (Puregon^®, MSD, France) were given on the 3^rd day of menstruation cycle and dose-adjusted according to the ovarian response. Later, GnRH antagonist (Orgalutran®, N.V. Organon, The Netherlands) was administered daily at 250 mcg when leading follicle reached 14 mm. A total of 250 mcg recombinant human chorionic gonadotropin (hCG) (Ovidrel®, Serono, Rockland, MA, USA) was given when at least three leading follicles or most of leading follicles in POR group reached more than 17 mm in diameter. Finally, OPU under transvaginal ultrasound guidance was performed at 36 h after hCG administration.

Follicular fluid and cumulus cell retrieval

The follicular fluid containing cumulus oocyte complex (COC) from the first puncture of dominant follicle was collected by OPU. The COCs were isolated from the follicular fluid. The follicular fluid was stored at -80°C while the COCs were cultured in Global^® for fertilization media (LifeGlobal, Guilford, CT, USA) containing 10% human serum albumin (LifeGlobal) at 37°C under 5% CO₂ for 2 h to 3 h. To separate the cumulus cells, the COCs were exposed to 70 IU hyaluronidase Type VIII (Sigma-Aldrich, St. Louis, MO, USA). The retrieved cumulus cells were washed in phosphate buffered saline and were stored at -80°C.

Evaluation of IL-6 level

IL-6 level in the follicular fluid was evaluated in duplicate using sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer protocol (IL-6 simple step ELISA® kit, Abcam, Cambridge, UK). Briefly, the standard curve with various concentrations ranging from 0 pg/mL (blank) to 500 pg/mL was generated. The optimal dilution of the follicular fluid was adjusted using the provided sample diluents. A total volume of 50 µL from each sample and antibody cocktail were added to 96-well plate and incubated at room temperature on a plate shaker (400 rpm) for 1 h. All samples were then washed. 3,3',5,5'-Tetramethylbenzidine substrate was directly added to each well, incubated at room temperature on the plate shaker (400 rpm) for 10 min and later by ELISA stop reaction. IL-6 level was measured using microplate spectrophotometer (Spectra Max M3 Multi-Mode Microplate Reader, Molecular Devices, CA, USA) through optical density at 450 nm.

Evaluation of TNF-α and GABRA-1 expression levels

Total mRNA from the cumulus cells were extracted using miRNeasy tissue/cells advanced micro kit (Qiagen GmbH). RNA concentration was then measured by NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). The first-strand cDNA was synthesized. Primers were as follows:

TNF-α

Forward: 5´-ATCAGAGGGCCTGTACCTCAT-3´

Reverse: 5´-AGACTCGGCAAAGTCGAGATA-3´

NCBI reference sequence = NM_000594.4

GABRA-1

Forward: 5´-ATGATGGAGCTCGAGGCAAA-3´

Reverse: 5´-AGCTCTGAATTGTGCTGGGT-3´

NCBI reference sequence = NM_000806.5

GAPDH

Forward: 5´-GGGGGAGCCAAAAGGGTCATCATCT-3´

Reverse: 5´-GAGGGGCCATCCACAGTCTTC-3´

NCBI reference sequence = NM_001357943.2

To evaluate expression levels of mRNA target, the relative quantitative polymerase chain reaction (qPCR) method was performed in duplicate using SYBR gene expression assay KAPA SYBR^® FAST qPCR Master Mix (2X) Kit on One Plus Real-Time PCR Machine (Applied Biosystem™, San Francisco, CA, USA). Each reaction was incubated under thermal cycling condition: initial denaturation at 95°C for 3 min, followed by 40 cycles of 3 sec at 95°C and later with either 20 sec at individually annealing temperature of each primer. For quantification, all of mRNA samples were normalized with GAPDH. The relative expression level was calculated using 2^-ΔΔCt method.

Statistical analysis

The data analyses were performed using IBM SPSS Statistics (Version 28). The independent variables included demographic data, IL-6 and gene expression and levels. The differences in dependent variable between experimental groups were compared using Mann-Whitney U test. In addition, the relation between two designed variables was carried out using Pearson’s correlation. All results are presented as mean ± S.D. Level of significance was set at P ≤ 0.05.

Demographic data

As demonstrated in Table 1, the demographic data did not reveal statistically significant differences in the age of patients or their partners between two groups. Average age for NOR group was 36.5 years old and POR was 37.4 years old, indicating slightly advanced age in POR group. In addition, body mass index did not demonstrate statistical significance. Regarding the medical record, the cause of infertility or the previous treatments such as intrauterine insemination and IVF cycles did not differ between groups.

Hormonal profiles and ovarian stimulation outcomes

Baseline hormonal profiles including FSH, (luteinizing hormone; LH) and estradiol did not differ between groups (Table 2). However, AMH was considerably lower in POR group (0.8 pg/mL compared to 2.2 pg/mL in NOR group) (P < 0.001). On the day of hCG administration, estradiol level in NOR group were higher (2,317.2 ng/mL) than those in POR group (1,153.1 ng/mL) (P < 0.001). For average AFC, the result showed significant reduction in POR (6.0 follicles) compared to NOR group (12.3 follicles) (P < 0.001) (Table 2).

According to the ovarian stimulation outcomes, this disparity was mirrored in the numbers of oocyte retrievals, which NOR group had significantly higher retrieved oocytes (11.4 oocytes) compared to POR group (3.6 oocytes) (P < 0.001). However, the maturation and the fertilization rates did not differ between patient cohorts (Table 2).

IL-6 and relative gene expression levels

IL-6 levels measured in the follicular fluid were slightly higher in POR group (0.016 ± 0.014 pg/mL) compared to NOR group (0.02 ± 0.01 pg/mL), however, the present data not reach statistical significance (P = 0.604) (Table 3).

Investigation of TNF-α expression levels in the cumulus cells demonstrated distinct differences between patients with NOR and POR. TNF-α showed notable disparity, with NOR patients exhibited higher expression level compared to those in POR group (P < 0.001). Conversely, GABRA-1 expression levels in the cumulus cells did not exhibit significant difference between two cohorts (P = 0.573) (Table 3).

Correlation analysis

As present in Table 4, the positive correlation of TNF-α in the cumulus cells to the baseline characteristics and the ovarian stimulation outcomes were observed; AMH (r² = 0.699), estradiol level on hCG day (0.611), AFC (r² = 0.731) and the number of retrieved oocytes (r² = 0.743), respectively. However, IL-6 and GABRA-1 expression levels did not present any correlations to all parameters.

Our present study mainly reported levels of pro-inflammatory (IL-6 and TNF-α) and cell apoptosis (GABRA-1) markers in the follicular fluid and the cumulus cells retrieved from the patients who normally and poorly responded to the ovarian stimulation during infertility treatment by IVF program. The major findings indicated that TNF-α expression levels in the cumulus cells retrieved from POR patients were higher than NOR patients. Besides, TNF-α level was positively correlated with the ovarian response, including levels of AMH, estradiol on day of hCG administration, AFC and the number of retrieved oocytes. However, IL-6 levels in the follicular fluid and GABRA-1 expression in the cumulus cells did not remarkedly differ between cohorts.

In general, IL-6 could have either pro-inflammatory or anti-inflammatory effects in different physiological micro-environments, leading to the controversy results on cell functions¹⁷. In terms of fertility outcomes, the previous study demonstrated that low levels of IL-6 (< 4 pg/mL) in the follicular fluid retrieved from > 14 mm follicles were associated with high pregnancy rate after embryo transfer¹⁸. In addition, high levels of IL-6 negatively affected oocyte quality in the patients with obesity¹⁹, endometriosis, or other chronic inflammation diseases²⁰. However, another study demonstrated that NOR patients who achieved the clinical pregnancy outcomes presented two-times higher of the follicular fluid IL-6 levels compared to non-pregnant group¹⁷. In addition, any negative correlations were not investigated regarding high levels of IL-6, the numbers of oocyte retrievals, and oocyte quality¹⁷. In our study, NOR and POR patients with normal BMI, age-matched without other inflammatory conditions were recruited. Furthermore, percentages of oocyte quality (metaphase II oocyte) from retrieved follicles did not differ between patient groups which might refer to comparable milieu. Thus, our present data possibly indicated that IL-6 might not interfere with the ovarian response during the ovarian stimulation program in terms of healthy (NOR) or POR patients who did not present any underlying inflammatory diseases.

In human ovary, expression of TNF-α and its receptor were observed in the oocytes and the cumulus cells of large antral follicles²¹. Moreover, TNF-α localization was also observed in other mammalian species, such as rat oocytes²². The activation of TNF-α receptor signaling was involved with the various adapter proteins called complex I and complex II²³. Complex I proteins consisted of TNFR1-associated death domain protein, TNF-associated factor 2, and receptor with protein 1²³. Complex II was formed by activation of complex I plus Fas-associated death domain and pro-caspase protein which played major role in cell apoptosis²³. The previous study in mouse model indicated that those two complexes were considered key mechanisms regulating granulosa cell apoptosis leading to follicular atresia²⁴. Another study revealed that TNF-α complex interfered with follicle development through nuclear factor kappa B and c-Jun N-terminal kinase pathways²⁵which were important in mediating cellular stress and apoptosis within ovarian milieu²⁶. The recent study demonstrated that in vitro treatment of resveratrol in cell culture generated from premature ovarian insufficiency could decrease TNF-α expression levels, leading to inhibition of premature depletion of ovarian follicles or folliculogenesis arrest in mice²⁷. Until now, TNF- α mechanism related to ovarian function has not been fully understood. However, the previous supporting data could possibly highlight paracrine effect of TNF-α on diminished follicle development which might subsequently be acknowledged as one of crucial roles in POR pathology.

For GABRA-1 levels, this is the first study that could explore expression of GABRA-1 mRNA in human cumulus cells. However, distinct patterns GABRA-1 expression levels between cohorts were not observed by our research. The previous study revealed that down-steam function of GABRA-1 by non-coding RNA (miRNA-139) successfully inhibited malignant cell proliferation in central nervous system¹⁶. Recently, cell apoptosis or cell proliferation suppression were approved as one of pivotal pathways to regulate the oocyte/cumulus cell quality in the IVF patients²⁸. When similar quality of oocytes from diminished the ovarian reserve patients and the healthy controls were compared, higher expression levels of capase-3 and cytochrome c were observed in the cumulus cells retrieved from the diminished ovarian reserve patients, indicating cell apoptosis event²⁸. According to our contradictory results, GABRA-1 might not be involved with the cellular pathways of the cumulus cell apoptosis related to poor follicle development in POR patients.

This study primarily demonstrated the differences of candidate inflammatory (IL-6 and TNF-α) and cell apoptosis (GABRA-1) markers in the IVF patients with normal and poor ovarian responses. In poor ovarian response cohorts, notably elevated levels of TNF-α were detected and present the positive correlation with AMH, number of antral follicle count, estradiol (on day of hCG treatment) levels, and number of retrieved oocytes. However, our research presented limitation on analyzing the cumulus cells and the follicular fluid from only one dominant follicle, which might not fully represent the cytokine profiles of all follicles from each individual patient. To address future research directions, expanding studies on TNF-α and other cytokines from all follicles retrieved from the ovarian stimulation will possibly improve knowledge on POR pathology and predictive accuracy of the ovarian stimulation outcomes.

AFC Antral follicle count

AMH Anti-müllerian hormone

COC Cumulus oocyte complex

FSH Follicle stimulating hormone

GnRH Gonadotrophin-releasing hormone

hCG Human chorionic gonadotropin

IL-6 Interleukin-6

IUI Intrauterine insemination

IVF In vitro fertilization

LH Luteinizing hormone

NOR Normal ovarian response

OPU Oocyte pick up

POR Poor ovarian response

rFSH Recombinant follicle stimulating hormone

Acknowledgments

The authors would like to sincerely thank all participants, Reproductive Biology Unit Staff (the medical fellows, the nurse and the nurse assistant for blood collection and oocyte pick up procedure as well as the embryologists for the follicular fluid and the cumulus cell collection).

Author contributions

PT and PS provided the study concept and design. PT and SS collected the biological samples. KR, PT, ST performed the research experiment, the clinical data collection, the analysis, and the interpretation under VS and WS guidance. KR initially prepared the draft manuscript while PT and PS revised and provided critique. The draft manuscript was approved by all authors.

Funding

This study was financially supported by Program Management Unit for Human Resources & Institutional Development, Research and Innovation (PMU-B), Office of National Higher Education Science Research and Innovation Policy Council, Thailand.

Competing interests

None of the authors disclose any conflict of interest.

Data Availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Table 1

Baseline characteristics of participants who underwent in vitro fertilization with normal ovarian response (n = 16) and poor ovarian response (n = 11) to ovarian stimulation (mean ± SD or %)
Baseline characteristic	Normal ovarian response	Poor ovarian response	P-value
Patient age (years)	36.5 ± 1.8	37.4 ± 2.9	0.355
BMI (kg/m²)	21.4 ± 2.0	23.3 ± 3.6	0.062
Partner age (years)	38.3 ± 4.8	37.8 ± 3.2	0.873
Infertility (%) Primary Secondary	85.7 14.3	81.8 18.2	- -
Cause of infertility (%) Unexplained Tubal factor Male factor	78.5 14.2 9.3	63.6 18.2 18.2	- - -
Previous treatment IUI (cycles)* IVF (cycles) *	1.8 ± 1.4 0.3 ± 0.5	1.5 ± 2.0 0.8 ± 0.6	0.450 0.090
* IUI = Intrauterine insemination; IVF = In vitro fertilization

Table 2

Baseline of hormonal profiles, antral follicle counts, oocyte retrieval, oocyte maturation rate and fertilization outcomes from participants with normal ovarian response (n = 16) and poor ovarian response to ovarian stimulation (n = 11) (mean ± SD or %)
Parameter	Normal ovarian response	Poor ovarian response	P-value
FSH (IU/L)*	6.6 ± 0.7	7.8 ± 2.5	0.144
LH (IU/L)*	5.7 ± 1.8	5.1 ± 1.6	0.691
Estradiol (ng/mL)	43.6 ± 20.1	41.6 ± 18.1	0.742
AMH (pg/mL)*	2.2 ± 0.4	0.8 ± 0.5	< 0.001
Antral follicle count	12.3 ± 2.4	6.0 ± 1.5	< 0.001
Duration of stimulation (day)	9.9 ± 0.9	9.2 ± 0.6	0.249
Estradiol level (ng/mL) on hCG* day	2,317.2 ± 453.3	1,153.1 ± 410.1	< 0.001
Number of retrieved oocytes	11.4 ± 2.1	3.6 ± 0.7	< 0.001
Maturation rate (%)	93.7 ± 4.6	87.7 ± 17.3	0.544
Fertilization rate (%)	94.1 ± 7.1	89.3 ± 11.5	0.162
* FSH = Follicle stimulating hormone; LH = Luteinizing hormone; AMH = anti-müllerian hormone; hCG = human chorionic gonadotropin

Table 3

IL-6 level in follicular fluid, relative expression levels of TNF-α and GABRA-1 in cumulus cells obtained from participants with normal ovarian response (n = 16) and poor ovarian response (n = 11) to ovarian stimulation (mean ± SD)
Candidate biomarker	Normal ovarian response	Poor ovarian response	P-value
IL-6 (pg/mL)	0.02 ± 0.01	0.016 ± 0.014	0.604
TNF-α	2.4 ± 1.5	7.1 ± 1.9	< 0.001
GABRA-1	1.9 ± 1.7	1.6 ± 1.1	0.573

Table 4

Correlation between TNF-α and selected parameters (baseline characteristics, IL-6 and GABRA-1 expression levels) obtained from participants with normal ovarian response (n = 16) and poor ovarian response to ovarian stimulation (n = 11)
Parameter I	Parameter II	r²	P-value
TNF-α	AMH	0.699	0.008
TNF-α	Estradiol level on hCG* day	0.661	0.010
TNF-α	Antral follicle count	0.731	< 0.001
TNF-α	Number of retrieved oocytes	0.743	< 0.001
TNF-α	Maturation rate	0.247	0.375
TNF-α	Fertilization rate	0.257	0.355
TNF-α	IL-6	0.062	0.802
TNF-α	GABRA-1	0.032	0.904
* hCG = human chorionic gonadotropin

No competing interests reported.

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Differences of pro-inflammatory and cell apoptosis markers between patients with normal and poor ovarian response during in vitro fertilization program Running Titel: Pro-inflammatory markers in IVF patients

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