Background: Only a few studies studies reported that granulin might provide various reproductive functions in the known literature. We aimed to investigate association of serum granulin levels with diminished ovarian reserve status.

Study design: This prospective cross-sectional study included a total of 111 infertile women admitting for fertility problems. The women were categorized into two groups according to their ovarian reserve statuses; 1-Normal ovarian reserve group (NOR, control group, n=72), and 2-Diminished ovarian reserve (DOR, n=39) group. The initial examination included the measurements of weight, height, waist circumference (WC) and hip circumference (HC) to calculate body mass index (BMI), and waist/hip ratio (WHR).

Results: There was no significant difference between both groups for the anthropometric measurements, including BMI, WC, HC and WHR (p>0.05, for all). Although the women with DOR had higher FSH levels (p<0.001), serum AMH concentrations were higher in the control group (p<0.001). The mean granulin level did not differ significantly in DOR and the control groups (p=0.229). The serum granulin levels in the normal ovarian reserve group correlated positively and significantly with BMI, WC and HC (r=0.320 and p=0.006, r=0.257 and p=0.029, and r=0.243 and p=0.040, respectively). No correlation between granulin and all study parameters in women with DOR was demonstrated.

Conclusion: There is no relationship of serum granulin level with DOR. Although the granulin level is correlated with anthropometric measurements including BMI, WC and HC in women with NOR, those correlations do not exist in women with DOR.

Granulin, Progranulin, Growth factor, Diminished, Ovarian reserve.

Background: Only a few studies studies reported that granulin might provide various reproductive functions in the known literature. We aimed to investigate association of serum granulin levels with diminished ovarian reserve status.

Study design: This prospective cross-sectional study included a total of 111 infertile women admitting for fertility problems. The women were categorized into two groups according to their ovarian reserve statuses; 1-Normal ovarian reserve group (NOR, control group, n=72), and 2-Diminished ovarian reserve (DOR, n=39) group. The initial examination included the measurements of weight, height, waist circumference (WC) and hip circumference (HC) to calculate body mass index (BMI), and waist/hip ratio (WHR).

Results: There was no significant difference between both groups for the anthropometric measurements, including BMI, WC, HC and WHR (p>0.05, for all). Although the women with DOR had higher FSH levels (p<0.001), serum AMH concentrations were higher in the control group (p<0.001). The mean granulin level did not differ significantly in DOR and the control groups (p=0.229). The serum granulin levels in the normal ovarian reserve group correlated positively and significantly with BMI, WC and HC (r=0.320 and p=0.006, r=0.257 and p=0.029, and r=0.243 and p=0.040, respectively). No correlation between granulin and all study parameters in women with DOR was demonstrated.

Conclusion: There is no relationship of serum granulin level with DOR. Although the granulin level is correlated with anthropometric measurements including BMI, WC and HC in women with NOR, those correlations do not exist in women with DOR.

Granulin, Progranulin, Growth factor, Diminished, Ovarian reserve.

Ovarian ageing is revealed by diminished ovarian reserve (DOR), and it has been blamed for age-related decline in fertility [1,2], increase in adverse reproductive outcomes such as miscarriages [3], and aneuploid gestations [4]. Ovarian ageing is one of the main factors that influences the outcome of in-vitro fertilization (IVF). Females with DOR usually respond to controlled ovarian stimulation poorly. This results in production of fewer oocytes, poorer quality embryos, and hence reduced implantation and pregnancy rates. The incidence of poor ovarian response, as a measure of reduced ovarian reserve, has been reported between 9 and 24% [5]. However, this rate tends to increase due to overall rise in the number of women that postpone conceiving to their 30s or 40s [6].

Granulin is also known as progranulin, acrogranin, proepithelin, and PC cell–derived growth factor. It is a secreted protein, and it has important functions in various processes, including immune response and development of embryo [7]. Granulin is a glycoprotein consisting of 593 amino acids, and its mRNA is expressed in a number of epithelial cells both in vitro and in vivo. Autosomal dominant mutations of progranulin gene result in frontotemporal dementia [8,9], On the other hand, overexpression of granulin helps invasive progression of some tumors, including breast and brain neoplasms [10]. It was reported that granulin overexpression caused resistance of breast cancer cells to tamoxifen [11]. This suggests that autocrine cancercell regulation of progranulin plays a significant role in tumorigenesis of breast cancer [12]. It has also been reported that progranulin mRNA is expressed in the inflammatory infiltrate in transcutaneous murine puncture wounds, and it is highly induced in fibroblasts of skin and endothelium after injury [13]. Granulin also increases accumulation of neutrophils, macrophages, blood vessels, and fibroblasts in cutaneous wounds [14], and this suggests that it may act as a chemotactic protein for myeloid originated cell types, and a factor of angiogenesis.

Only a few studies up to date have investigated associations of granulin with different ovarian reserve patterns. In this study, we aimed to investigate associations of serum granulin levels with DOR status.

The women included into the current study were the patients that admitted to the Reproductive Endocrinology Department of Hitit University Hospital for fertility problems between January and September 2016. This prospective cross-sectional study was approved by the Institutional Review Board of Ankara Numune Education and Research Hospital in accordance with the Declaration of Helsinki, 2013 Brazil version (E-15-563). All participants provided their written informed consents prior to the study. The exclusion criteria were history of pelvic surgery, chemotherapy and/or radiotherapy, ovarian masses, endometriosis, severe systemic diseases including diabetes mellitus, cardiovascular, renal, hepatic, autoimmune and endocrine disorders, use of drugs that are likely to affect ovarian functions, smoking, alcohol consumption, pregnancy and lactation.

A total of 111 infertile women who met the inclusion criteria were included in this study. The women were categorized into two main groups in relation with their ovarian reserve statuses. 72 women were classified as the normal ovarian reserve group (NOR, control group), and 39 consecutive women were diagnosed as diminished ovarian reserve (DOR) group. The diagnosis of DOR was based on Bologna criteria of ESHRE consensus [15]. At least two of following three characteristics are needed to diagnose the patient as DOR; (i) advanced maternal age (≥ 40 years) or any other risk factor for poor ovarian response (POR); (ii) a previous POR (≤ 3 oocytes with a conventional stimulation protocol; (iii) an abnormal ovarian reserve test (i.e. antral follicle counting < 5-7 follicles or antimullerian hormone <0.5-1.1 ng/dL). We missed the antimullerian hormone item for the study process.

The initial examination of women included the measurements of weight, height, waist circumference (WC) and hip circumference (HC) to calculate body mass index (BMI), and waist/hip ratio (WHR). BMI was calculated as weight (kg) / height2 (m2). WC was measured at the midpoint of the lowest margin of 12th rib and the lateral iliac crest during normal expiration. HC was measured at the maximum extension of the major trochantaries. All anthropometric measurements were made with the same scale, and by the same observer.

Venous blood samples were obtained from the antecubital veins in all participants in the morning, between 08 AM and 10 AM, after an eight-hour fasting period, on menstrual days 2–5 for estradiol (E2) and follicle stimulating hormone (FSH), luteinizing hormone (LH), antimullerian hormone (AMH) and granulin. The samples were allowed to clot completely at room temperature, and then they were centrifuged within 30 min at 3000 rpm for 20 min. The serums for AMH and granulin measurements were frozen at -80°C until analysis within 2 h of collecting the blood samples. The serums were analyzed on a daily basis for E2, FSH, LH, using electrochemiluminescence immunoassay (ECLIA) method with an auto-analyzer (Cobas 6000, E 601 Roche Diagnostics, GmbH, Mannheim, Germany). The AMH samples were measured by the ECLIA method with an autoanalyzer (Cobas 6000, E 601 Roche Diagnostics, GmbH, Mannheim, Germany). The granulin levels were measured using enzyme linked immunosorbent assay (ELISA) method and granulin assay kits (Biotek Synergy HT, Cloud-Clone Corp., Houston, USA). The comparisons and correlation analyses of all data were performed.

All data analyses were performed using SPSS (Statistical Packages for The Social Sciences) software, version 21 (SPSS Inc. Chicago, USA). The Kolmogorov-Smirnov test was used to test normality of distribution. Continuous parameters were presented as mean ± standard deviation (SD). Continuous variables were compared using independent samples t-test due to normally distributed. Spearman correlation was used to test whether Granulin showed any significant linear relationship with other study parameters. A p value less than 0.05 was considered as statistically significant.

The anthropometric and biochemical parameters in DOR and NOR groups are demonstrated in Table 1. As expected, the mean age of DOR group was higher than the mean age of the control group (p<0.001). There was no significant difference between two groups for the anthropometric measurements, including BMI, WC, HC and WHR (p>0.05, for all). The serum E2 and LH levels were similar in all women (p=0.070 and p=0.061, respectively). Although the women with DOR had higher FSH levels (p<0.001), serum AMH concentrations were higher in the control group (p<0.001). The mean granulin level did not differ significantly in DOR and the control groups (p=0.229).

correlation analysis revealed that the serum granulin levels in the normal ovarian reserve group correlated positively and significantly with BMI, WC and HC (r=0.320 and p=0.006, r=0.257 and p=0.029, and r=0.243 and p=0.040, respectively), as shown in Table 2. However, we did not observe any correlation between granulin and all study parameters in women with DOR (Table 2).

Our results showed that serum granulin levels did not differ between women with NOR and DOR. Only a few studies investigated the role of granulin on reproduction in the known literature. Several studies reported that granulin might provide various reproductive functions [16,17]. Suzuki et al. have suggested that granulin is an oocyte-secreted growth factor in late phases of maturation, and may act as an autocrine stimulator for maturation of oocytes [16]. The authors also emphasized the role of growth differentiation factor-9, a highly specific oocytic molecule modulating growth and differentiation of the early ovarian follicles [18].

Granulin has a number of characteristics that play role in other reproduction processes. In an experimental study on monkeys, Vendola et al. reported that granulin may cause excessive early follicular growth, similar to androgens [19]. In fact, granulin and cystein-rich growth factors have been reported to contribute early ovarian follicle development in ovary [20,21]. Moreover, it was suggested that granulin might play a part in interaction of oocyte and sperm, since acrosome of spermatozoa contains progranulin [17].

In our study, although the granulin levels did not show any correlation with the age of the patients, it was considered that it might be involved in senescence, similar to what happens in plants and other animals, probably independent of aging. Granulin levels gradually decrease with aging in plants. The granulin levels of POF patients are lower than the healthy individuals [22]. Those findings and the results of Suzuki et al. showing oocyte-specific expression of the granulin precursor, Progranulin, made us to suggest that the level of Granulin may be a marker for senescence of the human ovary, rather than senescence of human life [16].

In a study by Ersoy et al. it was reported that granulin levels were significantly lower in the patients with premature ovarian failure (POF) compared to the control group. However, their study group was not identical with ours. The authors suggested that granulin could be a candidate biomolecule that could have a potential for providing understanding of POF pathogenesis [23].

Our study has some limitations. Firstly, the sample size is small. Therefore, nonsignificant associations of granulin with some variables could have been found statistically significant if the sample size was larger. Secondly, our study included infertile women, not a normal women population.

In conclusion, we did not find any relationship of serum granulin level with DOR. Although the granulin level is correlated with anthropometric measurements including BMI, WC and HC in women with NOR, those correlations do not exist in women with DOR. Further prospective larger studies are needed to investigate the role of granulin on ovarian reserve and reproductive functions.

Ethics approval and consent to participate: This prospective cross-sectional study was approved by the Institutional Review Board of Ankara Numune Education and Research Hospital (E-15-563).

All participants provided their written informed consents prior to the study.

All data generated or analyzed during this study are included in this published article.

The authors declare that they have no competing interests.

This research was supported by the Scientific Research Unit of Hitit University, Corum, Turkey (TIP19001.14.005).

Umit Gorkem designed and performed the study. F Kerim Kucukler analyzed and interpreted the patient data. Cihan Togrul performed the statistical analysis. Nafiye Yılmaz supervised the study. All authors read and approved the final manuscript.

The authors acknowledge the staff of Scientific Research Unit of Hitit University, Corum, Turkey.