Follicular fluid levels of anti-Müllerian hormone, insulin-like growth factor 1 and leptin in women with fertility disorders

ABSTRACT

Anti-Müllerian hormone (AMH), insulin-like growth factor 1 (IGF1) and leptin are produced in the granulosa cells of follicles and play an important role in the growth and maturation of follicles. The aim of our study was to monitor AMH, IGF1 and leptin levels in a group of healthy women and compare them to a group of women with fertility disorders. The second aim was the evaluation of biomarker levels in relation to the identified cause of infertility. Totally, 146 females were enrolled into our study. Seventy-two healthy controls and seventy-four females with fertility disorders were divided into four subgroups: anovulation, endometriosis, fallopian tube damage, unknown reason. IGF1 was the only biomarker with significantly lower levels throughout the entire group with fertility disorders. We did not identify any statistically significant differences for AMH and leptin. Regarding subgroups, significant differences were only observed in the group of anovulatory women. AMH and leptin showed higher levels while IGF1 showed lower levels. In conclusion, levels of AMH, IGF1 and leptin found in follicular fluid are sensitive markers for anovulatory fertility disorders. AMH, IGF1 and leptin levels in follicular fluid have no relation to the fertility disorders caused by endometriosis, fallopian tube damage or disorders with unknown etiology.

Abbreviations: AMH: anti-Müllerian hormone; IGF1: insulin-like growth factor 1; PCOS: polycystic ovary syndrome

KEYWORDS:

• Follicular fluid
• fertility disorders
• anti-Müllerian hormone
• insulin-like growth factor 1
• leptin

Introduction

Currently, 10–15% of couples at reproductive age suffer from infertility. A better understanding of the composition of the medium in which the oocyte develops coupled with a better understanding of the regulatory processes of reproduction allow for the continuous improvement in the success of infertility treatment (Thoma et al. 2013). Anti-Müllerian hormone (AMH), insulin-like growth factor 1 (IGF1) and leptin are produced in the granulosa cells of follicles and both play an important role in controlling or influencing the growth and maturation of follicles (Khalid et al. 2000; Hamm et al. 2004; La Marca et al. 2006). Levels of biomarkers in the follicular fluid reflect the quality of the oocyte and its fertilization along with subsequent embryo development (Chen et al. 2016).

The aim of our study was to monitor the levels of select biomarkers in the follicular fluid in a group of healthy women compared to a group of women with fertility disorders. The second aim was a detailed evaluation of the biomarker levels in relation to the identified reason for infertility. We monitored the following biomarkers: AMH, IGF1 and leptin.

Results and discussion

AMH, IGF1 and leptin in follicular fluid have been monitored in many studies. Most of these studies observed individual markers in connection with individual disorders. We decided to monitor all three biomarkers for different groups of women according to the diagnosed reason of infertility. The first step of our evaluation was to test the age dependence of the assessed biomarkers. It is well known that AMH and IGF1 serum levels change during the course of life (Kelsey et al. 2011; Friedrich et al. 2014). In the case of leptin, changes with age have not been confirmed. We tested the age dependence of follicular fluid levels in a group of healthy women because biomarker levels in a healthy group are not affected by the presence of a fertility disorder. We divided the healthy controls into subgroups according to the following age groups: less than 20, 20–29, 30–39. No changes in any of the biomarkers in follicular fluid (Table 1) were found to be statistically significant (p = 0.6549, p = 0.2180 and p = 0.4748, respectively), thus age adjustment was not applied. Once we had clarified the relation of all biomarkers to age, we continued with the evaluation of individual biomarkers.

Table 1. Age dependency evaluation of biomarkers in the group of healthy controls in time periods of 10 years.

		p-Value Kruskal–Wallis test
Age	Count	AMH	IGF1	Leptin
<20	3	0.6549	0.2180	0.4748
20–29	46
30–39	23

AMH is produced in the granulosa cells of ovarian follicles (Kucera et al. 2016). AMH inhibits selection of primordial follicles and inhibits growth stimulation of preantral and antral follicles (Visser et al. 2006). AMH production starts in the follicles at a size less than 1 mm. The peak of production is reached in follicles of size 5–8 mm. In the large follicles of more than 8 mm, the production rapidly declines. AMH production within the follicles is the part of the mechanism responsible for selection of the pre-ovulatory follicle (Jeppesen et al. 2013).

Our findings fully agree with the mechanism described above. We observed significantly higher AMH levels (Table 2) in the group of anovulatory women (p = 0.0042) which is a sign of defective follicular development. AMH levels were very similar in all groups of women except the anovulatory group. We retrieved the follicles of range 16–20 mm. Usually, AMH levels decline with follicle growth. However, this mechanism is disrupted in anovulatory women. High levels of AMH disturb the ovulation process and disrupt the natural course of follicle maturation and selection of the pre-ovulatory follicle.

IGF1 serves as an intra ovarian regulator of granulosa cell function. IGF1 influences the granulosa cells through the autocrine mechanism and in cooperation with gonadotropins, IGF1 modulates the growth and development of the follicles (Mehta et al. 2013). In our study, IGF1 was the only biomarker which reached significantly lower follicular fluid levels in the group of women with decreased fertility compared to the group of healthy controls (p = 0.0088). The results are shown in Table 3. After the division of the group of women with decreased fertility into subgroups according to the types of fertility disorder (Table 2), we can see that lower levels of IGF1 were present in each subgroup in comparison with the group of healthy women. However, we only confirmed a statistically significant difference in the group of anovulatory women (p = 0.0036). Our results support the theory that IGF1 is an autocrine growth stimulator of follicles and plays a key role at different stages of follicular development. IGF1 is not required for primordial to primary follicle transition (Silva et al. 2009). However, IGF1 is necessary for the development of small antral follicles at the gonadotropin-dependent stages. IGF1 increases granulosa cell proliferation, steroidogenesis and oocyte growth (Yu et al. 2012). Anovulation caused the growth and maturation of follicles to stop. We found decreased levels of IGF1 in anovulatory women which suggests that the growth effect of IGF1 is lacking. Nevertheless, the precise mechanism of follicular growth and maturation is not yet fully elucidated.

Table 2. Biomarkers in females with reduced fertility according to the identified reason.

Status	N	Biomarker (ng/ml)	Mean	Median	Minimum	Maximum	p-Value Wilcoxon test
Anovulation	39	AMH	4.32	3.28	0.45	16.69	0.0042
		IGF1	105.6	97.5	53.0	236.3	0.0123
		Leptin	2486	2278	1283	6344	0.0089
Endometriosis	7	AMH	2.33	1.30	0.55	5.67	0.7245
		IGF1	108.7	113.0	60.5	157.0	0.3327
		Leptin	1084	802	403	2859	0.4150
Fallopian tube damage	12	AMH	3.53	2.08	0.60	16.56	0.4340
		IGF1	113.9	110.6	38.9	218.4	0.2666
		Leptin	1396	1121	580	3811	0.8235
Unknown reason of infertility	16	AMH	1.85	1.39	0.90	5.03	0.4101
		IGF1	108.3	110.5	10.9	190.9	0.1538
		Leptin	1458	1208	708	3337	0.9572

Bold values are significantly different, p-value < 0.05.

Table 3. Biomarkers in the follicular fluid in the group of healthy controls and women with decreased fertility.

Biomarker (unit)	Status	Mean	Median	Lower quartile	Upper quartile	Min. Max.	p-Value Wilcoxon test
AMH (ng/ml)	Healthy controls	2.74	1.72	1.19	2.88	0.34 18.2	0.4903
	Fertility disorders	2.90	1.92	1.01	3.94	0.45 16.69
IGF1 (ng/ml)	Healthy controls	129.1	124.5	103.3	151.6	67.5 332.8	0.0036
	Fertility disorders	109.1	106.7	81.9	132.1	10.9 236.3
Leptin (pg/ml)	Healthy controls	1501	1228	594.1	2172	149 5014	0.0089
	Fertility disorders	1687	1302	695.6	2499	74.3 6578

Bold values are significantly, p-value < 0.05.

Leptin belongs to the adipokine family. Leptin is predominantly synthesized and secreted by adipocytes in the white adipose tissue but is also expressed in different tissues and cells, including granulosa cells in ovaries (Wertel et al. 2005). Leptin is a hormone with an important effect on the reproductive system due to its role in cell growth and maturation.

The literature concerning leptin levels are controversial. Certain authors reported lower leptin levels in follicular fluid in anovulatory women with polycystic ovary syndrome (PCOS) (Garruti et al. 2014). Others reported increased levels in cases of endometriosis or unexplained reasons for infertility. They suggested that peritoneal fluid leptin can originate from the follicular fluid (Gogacz et al. 2001; Demir et al. 2007). Our results do not support this theory. Compared to the healthy patients, we observed higher leptin levels in anovulatory patients but lower levels in all other cases of infertility (Table 2).

In conclusion, levels of AMH, IGF1 and leptin in follicular fluid are sensitive markers in cases of anovulatory fertility disorders. However, no relation was observed in case of fertility disorders caused by endometriosis or fallopian tube damage and neither are they useful markers in cases of fertility disorders of unknown etiology.

Materials and methods

Subjects

In total, 146 females were enrolled into our study in which 72 were healthy controls (median age of 26 years). These women were participants in the donor oocyte project. The group of patients contained 74 females with fertility disorders (median age of 32 years). The group of patients were divided into four subgroups according to the reason for infertility: anovulation, endometriosis, fallopian tube damage and unknown reason.

Follicular fluid was collected from the patients during oocyte retrieval in the process of infertility treatment. Follicular fluid from the donors of oocytes was collected at the time of oocyte retrieval after stimulation with gonadotropins. Pools of follicular fluid samples from each female were investigated, which means a mixture of all follicular fluid samples with no blood contamination. The levels of AMH, IGF1 and leptin were determined for each sample. The study was approved by the local research ethics committee of the University Hospital. Written informed consent was obtained from all female participants.

Methods

Biomarker levels were assayed using the following kits: chemiluminescent kit ACCESS AMH (Beckman Coulter, Brea, CA, USA), chemiluminescent kit LIAISON IGF1 (Diasorin, Saluggia, Italy), Quantikine ELISA Human Leptin kit (R&D Systems, Mineapolis, MN, USA).

Statistical analysis

All statistical analyses were performed using SAS 9.3 (Statistical Analysis Software release 9.2; SAS Institute Inc., Cary, NC, USA). Quantitative variables are reported as median, mean, lower and upper quartile, minimum and maximum. The Wilcoxon test was used to compare the levels of biomarkers in the group of healthy females with the groups of females with fertility disorders. The Kruskal–Wallis test was used to compare the distributions of each biomarker across the age categories.

Disclosure statement

There is no potential conflict of interest reported by the authors.

Additional information

Funding

This study was supported by the Ministry of Health, Czech Republic - conceptual development of research organization (Faculty Hospital in Pilsen - FNPl, 00669806).

Notes on contributors

Radek Kucera

Designed the experiments: VB, RK, ZU-G; performed the experiments: RK, VB, VK; analyzed the data: RK, VB; wrote the manuscript: RK, VB, OT; revised the manuscript: RK, ZU-G. All authors approved the manuscript.