Correlation between luteinizing hormone receptor gene expression in human granulosa cells with oocyte quality in poor responder patients undergoing in vitro fertilization: A cross-sectional study

Background: This study was performed to evaluate the role of luteinizing hormone (LH) and granulosa cell LH receptor (LH-R) in poor responder patients who underwent controlled ovarian stimulation. Expression levels of LH-R mRNA in granulosa cells was investigated and compared with oocyte morphology, oocyte maturity and fertilization rate. Methods: Granulosa cells were obtained from 30 patients who underwent in vitro fertilization (IVF) at Dr. Cipto Mangunkusumo Hospital, Jakarta. The patients were divided into two groups: group I (n=10) poor responders; and group II (n=20) non-poor responders. After the extraction of total RNA from granulosa cells, semi-quantitative RT-PCR was performed and the amount of LH-R mRNA was quantified. The relative values were calculated as the ratio of LH-R mRNA and actin beta mRNA. Statistical analysis was performed using Mann-Whitney test and Spearman correlation. Results: The relative value of LH-R mRNA was higher in group I compared with group II (27.37[0.00-28939.37] vs 0.00[0.00-7196.12]). Oocyte maturity (r=0.267) and morphology (r=0.267) in group I consistently showed a positive correlation with LH-R mRNA; in group II a negative correlation with LH-R mRNA was shown for oocyte maturity (r= -0.552) and morphology (r= -0.164). Group I had a positive correlation between LH-R expression with fertilization rate (r=0.430), and group II showed a negative correlation (r=-0.340). Conclusions: The expression of LH-R mRNA has a positive correlation with oocyte quality in poor responder patients and a negative correlation in non-poor responders. Our study suggests an optimal expression of LH- R mRNA in granulosa cells during controlled ovarian stimulation to obtain good quality oocytes.

non-poor responders. Our study suggests an optimal expression of LH-R mRNA in granulosa cells during controlled ovarian stimulation to obtain good quality oocytes.

Introduction
In 2012, the World Health Organization reported that 80 million reproductive-aged couples, which constitutes 10% of the total number of couples globally, have issues related to infertility 1 . In Indonesia, 12-15% of reproductive-aged couples have infertility issues 2,3 . One way to manage infertility issues is using assisted reproductive technology (ART). One method of ART that is commonly used is in vitro fertilization (IVF). The In Vitro Fertilization World Report 2000 showed that the average number of pregnancy and births post-IVF is 26.7% and 18.6%, respectively 4 . According to a 2008 report, the percentage of pregnancy post-IVF in Indonesia is 20-52.9% 5 . As reported by the Society of Assisted Reproductive Technology, the success rate of IVF in women under 35 years of age is 41-43% 6 . However, this success rate declines as a woman's age increases, especially for women who are older than 35 years old and those who are not adequately affected by gonadotropin treatment (also known as a poor responders) 7 . Many theories have tried to explain the etiology of poor ovarian response towards gonadotropin administration. Age and low ovarian reserve are the most common factors used to explain the presence of a poor responder patient group. However, some poor responders are still young, thus the etiology of poor responders seems to be multifactorial, including decreased blood flow to the ovary, decreased aromatase activity, FSH and LH receptor polymorphisms, and autoimmunity towards the ovary 26 . Understanding LH-R gene expression in humans is important to increase the success rate of IVF. The importance of LH during follicular phase and the optimum dosage of LH for IVF patients are still debatable 27 . Genetic studies have had important roles in understanding the pathogenesis of diseases and development of therapy. By understanding genetic studies with a focus on gene and gene product, IVF specialists can decide the appropriate therapy for patients 18 . Therefore, this study investigates the correlation between LH-R granulosa expression in poor responder patients and non-poor responder patients who are going through an IVF program and compares the oocyte quality outcome, fertilization rate, and pregnancy rate.

Study setting
This cross-sectional study was conducted to find the correlation between LH-receptor gene expression in granulosa cells with oocyte quality in poor responder patients undergoing IVF.

Participants
Women attending the Yasmin Clinic for IVF procedures were selected according to Bologna criteria based on anamnesis, ultrasound, and laboratory examination 8 . The patients underwent ovarian stimulation, which is part of the IVF procedure, continuing to the ovum pick-up (OPU) procedure. Prior to OPU, patients were offered to participate in the study. An explanation of the research, objectives, procedures, benefits, risks and expected study outcomes were provided, along with an informed consent form. The subjects who were willing to participate in the study were asked to sign the consent form. Patients with incomplete baseline data and who failed the OPU procedure were not included in this study. In total, 30 patients were recruited from January to June 2015 in the Yasmin IVF Clinic Dr. Cipto Mangunkusumo General Hospital, Jakarta. The patients were divided into two groups: I poor responders (n=10); and II non-poor responders (n=20). The poor responder group (group I) matched minimum 2 of these following criteria: (1) more than 40 years of age; (2) ovary reserve test such as the basal antral follicle count (BAF) <6-8 follicles/ovary, or level of anti-Müllerian hormone (AMH) <0.5-1.1 ng/mL; and (3) history of ovary stimulation producing < 3 oocytes. Patients who do not have those criteria, entered into group II.

Data collection
At the time of OPU, the doctor extracted intrafolicular fluid from the patient under anesthesia. The intrafolicular fluid containing oocytes and granulosa cells was processed by the embryologist, and then the oocyte is processed in the next stage of IVF. The granulosa cells were stored at -20°C before use.
The RNA of the granulosa cells was extracted using High Pure RNA Isolation Kit (Roche, Mannheim, Germany). Measurement of total RNA concentration was made using NanoVue spectrophotometer (General Electric). Subsequently, cDNA synthesis was performed. The positive control used was mRNA from the Transcriptor First Strand cDNA synthesis kit (Roche). A light cycler fast start DNA MasterPLUS SYBR green I (Roche) was used for real time polymerase chain reactions (PCR), with the following profile: pre-incubatiton (1 cycle at 95°C for 10 minutes), quantification (45 cycles each at 95°C for 10 seconds, 65°C for 10 seconds and 72°C for 25 seconds), melting curve (1 cycle each at 95°C for 0 seconds, 65°C for 60 seconds and 95°C for 0 seconds). All the procedures are according to the manufacturer's instruction.
In this study, we used one microgram of complementary DNA (cDNA) per reaction in a 10 microliter reaction volume. Betaactin RNA was chosen as a suitable nonpoorization control gene. LH-R gene quantification was done using Light Cycler Fast Start DNA MasterPLUS SYBR green I kit (Roche). The real time PCR was performed using Light-Cycler 2.0 Instrument (03531414001, Roche). Primer sequences can be seen in Table 1.
Output data of qRT-PCR were used to calculate the ratio of gene LH-R is the value of delta Rn. The value is the result of fluorescence detection by qRT-PCR machine and translated by using LightCycler Software Version 4.1 (Roche). The Rn value was stored in file comma delimited file (CSV) using the Kingsoft Spreadsheet program Version 2013. The data were then processed using the R Studio program Version 2.11.1, which had been added to the qpcR software package. The result of script application is the ratio value, Cp, and the efficiency of each reaction.
For the oocyte morphology data, we used modification scoring system based on the Xia criteria, including first polar body, perivitelline space and cytoplasmic granulation ( Table 2) 28 . Morphology scores were measured to all oocyte retrieved with minimum score of 0 and maximum scores of 6. Mean value of morphology scores per patient were used for analysis. The percentage of fertility rate were measured from the total of fertilized divided by total embryos.

Data analysis
Data of expression of LH-R gene, oocyte maturity, morphology, and fertilization rate were analyzed. The normality of the data was tested using Shapiro-Wilk test. Normally distributed data were then tested with the unpaired T-test, but if the data had an abnormal distribution, the Mann-Whitney test was performed to compare the differences in expression of LH-R gene between groups of poor responder and non-poor responder  Table 3 shows the characteristics of groups I and group II, poor and non-poor responders, respectively. We found that granu-losa LH-R expression is higher in the poor responders (27.37 (0.00-28939.37) arbitrary unit) than in non-poor responders (0.00 (0.00-7196.12)), but this was not statistically significant between the groups (p=0.169). Table 4 shows a statistically significant negative correlation between oocyte maturity in the poor responders and granulosa LH-R expression (p=0.003; r=0.552). There is no correlation between granulosa LH-R expression and oocyte morphology. There is negative correlation between granulosa LH-R expression

Discussion
From this study, granulosa LH-R expression in poor responders is higher than in non-poor responders, although it is not statistically significant. In a study by Thiruppathi et al. 29 , which compared gonadotrophin receptor expression in poor responders and non-poor responders, the results also showed that granulosa LH-R expression is higher in poor responders. This may suggest that there is a disruption in LH-R processing and trafficking or it could be caused by accelerated release of LH in poor responders 29 .
In this study, granulosa LH-R expression had a positive correlation with morphology, oocyte maturity and fertility rate in the poor responder group and a negative correlation in non-poor responder group. This showed that the poor responder group needs LH for oocyte growth and maturation, while in the non-poor responder group high granulosa LH-R expression would affect maturity and morphology of oocytes and fertility rate. Maman et al. 18 found that in non-poor responders, granulosa LH-R expression increased in the antral-phase follicle and the highest expression happened pre-ovulation. There was a correlation between LH-R expression and fertility output. In that study, low LH-R expression correlated with low oocyte maturity, but excessive LH-R expression correlated with a low fertility rate 18 . From these studies, it seems that an optimal granulosa LH-R expression is needed to mature oocyte and yield a good fertility rate.
A literature study by Shoham 19 suggested that there is a therapeutic window with a threshold and ceiling in LH supplementation. If the LH level is below the needed ceiling, estradiol production will not be adequate, while if the LH level is higher than the threshold, there will be a negative impact on follicle growth. Shoham discovered that LH supplementation in patients with hypogonadotropic hypogonadism would generate more follicles and adequate estradiol levels to generate good endometrium growth. However, excessive LH supplementation in patients with hypogonadotropic hypogonadism or polycystic ovaries would cause negative effects, causing follicles to become atretic 19 . The study is supported by Humaidan et al. 29 , who found that LH level must be at an adequate level, not too high or too low, to generate good quality oocytes. Optimal LH levels measured in the 8th day of stimulation will decrease the required FSH doses, hence making the stimulation duration shorter and growth of good follicles faster 29 . However, it must be remembered that this study was done in non-poor responder patients, using a GnRH agonist protocol, where patients' gonadotropin is stimulated with GnRH agonist administration before stimulation. Therefore, if endogenous LH levels decrease too much because of that suppression, follicular growth will be disrupted, because the androgen production in theca cells, which will be converted into estradiol in granulosa cells, is decreasing. Because of that, non-poor responder patients who receive an agonist GnRH protocol should use an adequate dose that is not too high. Besides the dose, the administration mode also influences mid-follicular LH levels.
In the administration of intranasal buserelin, the decrease of mid-follicular LH is not too low, and the pregnancy rate is better compared to subcutaneous administration 29 .
In the present research, all subjects received the antagonist protocol, so there was no excessive endogenous LH suppression as seen in the agonist protocol. It seems that the stimulation protocol type may not influence granulosa LH-R expression. This is supported by a microarray analysis of gene expression study in rFSH and hMG stimulation, where LH-R expression showed no differences 30 . Therefore, it may be inferred that the main contributors of granulosa LH-R expression are follicle size and follicle maturation stage 18 . Granulosa LH-R is expressed in the early antral phase of follicle growth even when granulosa LH-R expression is still very low 18,31 . Then, by increasing follicle maturation under FSH influence, granulosa LH-R expression will also increase. Therefore, granulosa LH-R expression can be increased by exposing granulosa to adequate FSH before 18 . However, how much FSH to get optimal LH-R expression must be investigated. The most common problem in poor responder patients, other than the inadequate number and bad quality of oocytes produced, is that the FSH dose needed for stimulation is too high. This has been shown in the present study (Table 2). In this study, the total FSH dose used in the poor responder group was higher than that in the nonpoor responder group (3660 IU vs 2822 IU). In the Alviggi study, a higher FSH dose was also found in polymorphism LH patients (v-betaLH). According to Alviggi, homozygote and heterozygote v-betaLH patients have a poor ovarian response to gonadotropin and the need of total FSH dose is higher than in wild-type LH patients. The oocytes are also generated in a smaller number. In his hypotheses, Alviggi stated that it was caused by the difference in the bioactive effects between v-betaLH and wild-type LH 25,32 . v-betaLH has shorter t-half but has a more potent efficacy at receptor level compared to the wild-type 25 . In the present study, analysis of v-beta LH was not done. Besides LH polymorphism, in the Alviggi study, there was also a patient who did not have v-betaLH but had an inadequate response to gonadotropin. The possible explanation of that phenomenon is due to the increment in FSH consumption without the presence of v-betaLH, which is caused by LH-R and FSHR polymorphisms 32 . However, this still needs to be further investigated.

Conclusions
This study showed that granulosa LH-R expression in poor responders is higher than non-poor responders. Statistical analysis showed a positive correlation between granulosa LH-R expression with oocyte quality and fertility rate in the poor responders, and a negative correlation between granulosa LH-R expression with oocyte quality and fertility rate in the nonpoor responders.

Data availability
Underlying 1.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Yanping Kuang
Reproductive Medicine Center, Shanghai Ninth Hospital, Shanghai Jiao Tong University, Shanghai, China The aim of this study was to evaluate the role of LH and granulosa cell LH-R in poor responder patients who underwent COS. Then the authors analyzed the expression of LH-R in the granular cells from poor responder and non-poor responders and found the expression of LH-R mRNA had a positive correlation with oocyte quality in poor responder patients and a negative correlation in non-poor responders. The aim of this study was vague. The conclusion was not consistent with the purpose of this study and made with insufficient supporting data. The correlations between the LH-R expression and oocyte quality was established based on Spearman's test analysis. However, the oocyte quality might be correlated with age, BMI, basal FSH level, basal LH level and other factors. To analyse the correlation between the expression of LH-R and oocyte quality, multivariate logistic regression should be performed to quantify the effect of all related factors on the oocyte quality.

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: I'm researcher in the field of reproduction and embryolog.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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