The relationship between H19 and parameters of ovarian reserve

In order to evaluate circulating H19 expression and correlation with ovarian reserve in infertile women, discarded blood samples (2 mL) were collected from 69 patients undergoing evaluation for infertility at the Yale Fertility Center (New Haven, CT) (Table 1). Samples were collected in the early follicular phase (day 2–4 of the menstrual cycle). Serum was separated by centrifugation for 10 m at 12000 rpm at 4 °C and stored at − 80 °C until RNA extraction [20‐22]. Patients were excluded if any of the following diagnoses were present: Cushing’s syndrome, hyperprolactinemia, adrenal hyperplasia, acromegaly, hypothalamic amenorrhea, hypothyroidism, or diabetes mellitus. Patients were divided into three groups: controls (women diagnosed with male and tubal factor infertility, n = 19), women with unexplained infertility (n = 24), and women with diminished ovarian reserve (DOR, defined as AMH < 1.1 ng/mL and antral follicle count < 5, n = 26). This study was approved by the Yale University Institutional Review Board (IRB protocol # 1606017946).

We also sought to determine whether H19 is detectable in cumulus cells of women undergoing IVF with oocyte retrieval, and whether expression correlates with ovarian reserve as measured by response to gonadotropins. For these studies, we utilized pooled cumulus cells collected from a total of 72 consecutive cycles in women undergoing infertility treatment with IVF-ICSI at Gazi University School of Medicine IVF Center (Ankara, Turkey) (Table 2). Causes of infertility included diminished ovarian reserve, male factor, tubal factor, anovulation, endometriosis, and unexplained infertility, or a combination of these factors. For patients undergoing agonist cycles, treatment was initiated by GnRH agonists during the luteal phase of the preceding cycle. Stimulation with gonadotropins was initiated after downregulation had been achieved (estradiol level < 50 pg/ml in the absence of ovarian cysts on transvaginal sonography). For patients undergoing antagonist cycles, treatment with gonadotropins was initiated on cycle day 3 when serum progesterone was < 1 ng/ml and transvaginal sonography confirmed absence of ovarian cysts. A GnRH antagonist was added for pituitary suppression after five days of gonadotropin stimulation. Stimulation protocols included 150–300 IU/day of gonadotropins, either recombinant (GonalF, Merck) or in combination with human menopausal gonadotropin (hMG, Menopur, Ferring). Patients received hCG (Ovidrel 250 μg, Merck) when two or more follicles > 18 mm in diameter were present with an adequate estradiol response. Oocytes were collected 36 h after hCG injection. Retrieved cumulus-oocyte complexes (COCs) were placed in culture medium (G-MOPS, VitroLife) and cumulus cells were dissected from the oocyte mechanically in the absence of hyaluronidase. Cumulus cells from each patient were pooled in a single eppendorf tube. Samples were washed twice with 0.5 ml 1X Phosphate Buffered Saline (PBS) and centrifuged at 1000 x g for 1 min with the supernatant removed after each wash. After the final wash, the cell pellet was resuspended in 50 μl SideStep™ Lysis and Stabilization Buffer (Stratagene, La Jolla, CA). Samples were stored at − 80 °C until analysis.

For study analysis, patients were stratified by percentiles based on the number of retrieved oocytes. The number of oocytes included in the first quartile, which correspond to percentile values lower than 25%, was considered as indication of poor response (PR) to controlled ovarian stimulation. Women producing more oocytes than the 75th % of their age group were considered high responders (HR) to controlled ovarian stimulation. Women whose oocyte production fell between 25-75th % of their age group were considered normal responders (NR). This study was approved by the Gazi University Institutional Review Board committee (IRB protocol #131/11.05.2011).

Primers for H19 and β-actin were purchased from Real Time Primers (Elkins Park, PA, USA). The Norgen Plasma/Serum RNA Extraction Kit was purchased from Norgen Biotek (Ontario, CA). The Qiagen miRNeasy Mini Kit and RNeasy MinElute Cleanup Kits were purchased from Qiagen (Germantown, MD, USA). SYBRGreen was purchased from BioRad Laboratories (Hercules, CA, USA).

RNA extraction from stored blood samples was performed using the Norgen Plasma/Serum RNA Purification Mini Kit (Thorold, Ontario, Canada). To purify RNA from cumulus cells, the Qiagen miRNeasy Mini Kit and RNeasy MinElute Cleanup Kits were used according to the manufacturer’s instructions. RNA quantity and purity were determined using an ultraviolet spectrophotometer. Reverse transcription was carried out using the BioRad iScript cDNA synthesis kit (Hercules, CA, USA) in a 20ul reaction containing 0.5μg of total RNA. For cDNA synthesis, the reaction mixtures were incubated at 16 °C for 30 min, at 42 °C for 30 min, and at then 85 °C for 5 min. The cDNA was stored at − 80 °C until qPCR. qPCR was performed in a 25ul reaction containing 1.5ul of cDNA using Bio-Rad QSYBRGreen in a Bio-Rad iCycler. PCR was performed by initial denaturation at 95 °C for 5 min followed by 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. All SYBR green runs had dissociation curves to predict potential primer-dimers. Specificity was verified by melting curve analysis and agarose gel electrophoresis. The threshold cycle (Ct) values of each sample were used in the post-PCR data analysis, and the delta-delta Ct method was used to calculate mRNA expression. H19 levels were normalized and expressed as fold change relative to that of β-actin. The mean level of H19 expression in the control (tubal/male) factor group was set to 1, and H19 expression in the other two groups was normalized to this control group. All SYBR green runs had dissociation curves to predict potential primer-dimers. Specificity was verified by melting curve analysis. The PCR primers for the indicated genes are listed below.

Human H19 forward: 5′-GCACCTTGGACATCTGGAGT.

Human H19 reverse: 5′-TTCTTTCCAGCCCTAGCTCA.

β-actin forward: AAGAGCTATGAGCTGCCTGA.

β-actin reverse: TACGGATGTCAACGTCACAC.

Descriptive statistics for all patients were analyzed with one-way ANOVA combined with Bonferroni post hoc analysis. To compare H19 expression levels, one-way ANOVA was performed with Bonferroni post hoc analysis. Association between serum H19 expression and AMH levels was measured using Pearson’s correlation coefficient analysis. All P values were considered to be statistically significant when p < 0.05. Statistical analysis was performed using GraphPad (Chicago, IL).

Women with DOR had significantly lower serum H19 expression level as compared to women with tubal factor infertility and women with unexplained infertility (Fig. 1a, p < 0.05). As expected, women with DOR also had significantly lower serum AMH levels 0.4 ng/mL for DOR vs 2.5 ng/mL for tubal factor and 3.0 ng/mL for unexplained infertility, p < 0.001). A moderate correlation was observed between serum H19 expression and AMH (Fig. 1b, p < 0.05).

H19 expression was significantly decreased (4.6 fold) in cumulus cells of IVF patients who demonstrated a poor response to gonadotropins compared to high responders (Fig. 2, p < 0.05). Women who were poor responders also had higher cycle day 3 FSH levels (8.6 mIU/mL vs 6.4 mIU/mL, p < 0.05) and lower peak E2 during IVF stimulation (1175.2 pg/mL vs 2094 pg/mL, p < 0.05). Total gonadotropin dose did not differ between the two groups.