Research Article
Eudoxia Mamas1, Despoina Mavrogianni2, Rami Raouasnte2, Spyros Karkatzoulis2, Emmanouela Liokari2, Petros Drakakis1, Dimitrios Loutradis1
11st Department of Obstetrics & Gynecology, Alexandra General Hospital, Medical School of National and Kapodistrian University of Athens, Athens, Greece, 2Molecular Biology Laboratory, IVF Unit, 1st Department of Obstetrics & Gynecology, Alexandra General Hospital, Medical School of National and Kapodistrian University of Athens, Athens, Greece
Correspondence: Eudoxia Mamas, 26, Riga Feraiou st, N. Psichiko, 15451, Athens, Greece, Tel.: +30 6932429800, e-mail: eudoxia.mamas@gmail.com
Abstract
Assisted Reproduction Technology (ART) has proven to be a valuable tool for infertile couples. Unfortunately, no perfect ovarian stimulation protocol has been designed and ovarian response shows great variability among women. Pharmacogenomics aims at detecting genetic markers so as to individualize protocols in order to maximize ovarian response to treatment. Follicle Stimulation hormone receptor (FSHR) mutations and single nucleotide polymorphisms have been extensively studied. Splice variants of the FSHR have been detected in women undergoing in vitro fertilisation (IVF). This study aims to determine the presence FSH splice variants in Greek women undergoing IVF. RNA was extracted from cumulus cells from 35 women and analysed by real time PCR. Splice variants were detected by gel electrophoresis. Three cases of deletion of exon 9 and 2 cases of insertion of intron 8 were detected in our study group. No association between the presence of splice variants and response to ovarian stimulation was detected. Two live births were detected one in each variant group. Even though these two types of splice variant detected do not show any clinical correlation it is believed that variants of the FSHR may be associated with poor or high response to exogenous gonadotrophin so further research is necessary.
Keywords: Follicle Stimulation Hormone Receptor (FSHR), splice variants, Assisted Reproduction Technology (ART), ovarian stimulation, ovarian response.
Introduction
FSH is one of the fundamental hormones of mammalian reproductionresponsible for folliculogenesis1 and spermatogenesis2. FSH is a member of the glycoprotein hormone family along with LH, TSH and hCG. Each of these hormones share a common α subunit and differ in their unique β subunit.FSH regulates ovarian and testicular function through binding with the FSH receptor located in granulosa3 and Sertoli cells4. The FSH receptor (FSHR) is a G protein-coupled receptor composed of seven transmembrane helices flanked by intracellular and extracellular domains. The intracellular portion which is coupled to a Gs protein, activates the adenyl cyclase enzyme causing an increase in intracellular cAMP and activation of the cAMP dependent protein kinase pathway5. Inactivating mutations of the FSHR gene have been identified in women with primaryor secondary amenorrhea, infertility, polycystic ovary syndrome whereas activating mutations have been linked with ovarian hyperstimulation syndrome6.
FSH is one of the key hormones used during ovulation induction. It has been noted however, that the ovarian response to FSH stimulation shows great interindividual variability. Many scientists have throughout the years attempted to identify factors responsible for this variable response. Numerous single nucleotide polymorphisms (SNPs) have been located in the FSHR gene,however only two amino acid substitutions in the coding region (p.307Thr/Ala rs6165 and p.68-Asn/Ser rs6166)have been extensively studied regarding theirrelationship with ovarian response and IVF outcome7,8.
Following cloning of the FSHR cDNA, it became apparent that alternatively spliced transcripts were co-expressed in the gonads of various species including the cattle, rat, mouse, sheep and human9,10. In humans, deletion of exons 6 and 9 and insertion of part of intron 8 has been identified in infertile men11. For women undergoing IVF, del exon 2 has been linked with poor ovarian response whereas, del exon 6 with a high ovarian response. Deletion of exon 9 and insertion of 102bp of intron 8 has also been reported in this group5.
Material and Methods
Patient selection and ovulation induction
Patients were recruited from the IVF clinic of Alexandra Hospital, Athens, Greece. The research protocol was approved by the Ethics Committee of Alexandra Hospital and all participants signed an informed consent before enrolling to the study.
Demographic characteristics (age, Body Mass Index, duration and cause of infertility) and baseline hormonal profile (day 2 FSH, LH and PRL) were recorded before ovarian stimulation.
Gonadotrophin dose, duration and response to ovarian stimulation, number of oocytes and fertilised oocytes were monitored during the treatment cycle for each patient.
Ovulation induction
For agonists cycles, treatment was initiated following pituitary suppression using intranasal Buserelin spray (Superfact; Hoechst, Frankfurt, Germany). Downregulation and resulting ovarian suppression was confirmed with E2 levels (<40 pg/ml) and ultrasound scan (absence of follicular cysts). In antagonist cycles, treatment was initiated on day 2 or 3 of the cycle after ultrasonographic evaluation revealed the absence of ovarian cysts. Stimulation with 150 – 300 IU/day of either recombinant FSH (Gonal-F, Serono, Geneva, Switzerland) or in combination with hMG (Merional, IBSA, Lugano, Switzerland) was then initiated. Serum E2 levels were measured on day 5 and then daily after day 8. Follicle growth and endometrial thickness was assessed daily by ultrasound from day 9. In cases of abnormal ovarian response the dose of rFSH was adjusted. When two or more follicles reached 18mm or greater in diameter with adequate serum E2 level increase, hCG (10,000IU, Pregnyl; Organon, Oss, Netherlands) was administered for triggering final oocyte maturation. Oocytes were collected 36 hours later by transvaginal ultrasound guided ovarian puncture.
RNA extraction and cDNA preparation
Following oocyte retrieval, oocytes were stripped of the surrounding cumulus oocyte complexes (COCs) with mechanical manipulation in hyaluronidase and COCs were collected by aspirating the hyaluronidase solution in a 1.5 ml tube and centrifuged at 1000 x g for 10 minutes. RNA was extracted using the RNeasy Micro Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. RNA was then reverse transcribed using 8µl dNTP mix, 10µl nuclease free water, 4µl oligo dT Primer (Ambion, Austin, TX, USA), 2µl ribonuclease inhibitor and 2µl M-MLV reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA). The reaction was carried out in Mastercycler (Eppendorf, Hamburg, Germany) using the following conditions: 80°C for 3 min, 42°C for 60min and 92°C for 10min. The cDNA products were then stored at -20°C. Quality of cDNA was evaluated by amplification of G6PD gene (LightMix Kit human G6PD, TIB MOLBIOL, Berlin, Germany) using the LightCycler 480 II (Roche, Basel, Switzerland).
Detection of splice variants with RT-PCR
cDNA was used for the detection of splice variants of the FSHR gene through reverse transcription PCR (RT-PCR). The primers for the primary PCR were obtained by TIB MOLBIOL as follows: FSHR.1F, 5’GGA GGT TTT TCT CTG CAA ATG CAG3’ and FSHR.2R, 5’CAT ACC CTT CAA AGG CAA GGA CTG3’. The RT-PCR program was 50°C for 30min, 95°C for 15min, 10 cycles: 92°C for 30sec, 65°C for 20sec, 72°C for 3min; 20 cycles: 92°C for 20sec, 55°C for 15sec 72°C for 5 min. Nested PCR for FSHR was performed using the same conditions and the combination of primers: FSHR.9F, 5’ATG GCC CTG CTC CTG GTC TC3’ with FSHR.10R, 5’CAA ACT CAG TGT ACG TCA TGT C3’ and FSHR.2F, 5’ ATC CCA GCC ATT GCT GTG CCT TTG3’ with FSHR.8R, 5’TTA GTT TTG GGC TAA ATG AC3’. The PCR products were analysed on 2% agarose gel.
Results
A total of 35women with infertility due to malefactor, tubal factor or unexplained infertility were included in the study. The women were of Greek origin, aged 28 – 40 years old with a normal basal serum FSH level (2 – 10IU/lt).
RNA was extracted from cumulus cells, cDNA was synthesized and G6PD expression was measured using real time PCR.Amplification of FSHR cDNA was then carried out using nested PCR. Transcripts of the receptor were detected30 out of 35 patients (85%). These samples were further analysed to assess the presence ofalternatively spliced variants of the FSHR.
Ovarian response to ovulation induction was determined by the number of retrieved oocytes and the oocyte maturation rate. Pregnancy rates were also recorded.
Splice variants were identified in 17% of the patients (5 of 30); three patients showing deletion of exon 9 (Figure 1) and two patients showing insertion of intron 8. Table 1 shows the baseline characteristics among the 30 women with normal FSHR transcripts and the 5 women
Figure 1. RT-PCR products from women showing deletion of exon 9 FSHR variant.
withFSHR splice variants. These five patients showed similar response to ovulation induction irrespective of the induction protocol used (two withagonist and three with antagonist protocol). All five patients showed a good ovarian response with more than 8 oocytes retrieved and a mean oocyte maturation rate of 81%. Two live births were recorded in the group of patients where splice variants were identified (Table 2).
Discussion
ART has proven to be a valuable option in couples suffering from infertility. Following extensive research it has been deduced that no definite (demographic, anthropometric, hormonal) characteristic can predict a woman’s ovarian response to exogenous gonadotrophins12.Some women show resistance to exogenous gonadotrophins also knownhyporesponse, a term included in the new POSEIDON classification of ‘low prognosis’ women undergoing ART13. This has urged researchers to look for variants that could affect ovarian response to stimulation ultimately allowing scientists to design patient specific ovarian stimulation protocols. This pharmacogenomic approach if applied in ART will ultimately minimise poor response rates or excessive ovarian response in ovulation induction protocols. The available research is however very limited proving that the field of pharmacogenomics in ART is still immature.
In this study, alternatively spliced variants of the FHSR were analysed in Greek women undergoing IVF treatment. Based on the limited available literature, we focused on locating deletion of exon 2, 6 and 9 as well as insertion of 102bp in intron 8 in this group of women5.
Of the FSHR splice variants investigated in this study, only two have been shown to have clinical significance in women undergoing IVF in North America and Turkey. Deletion of exon 2 has been linked to poor ovarian response to exogenous gonadotrophins. This FSHR variant when co-expressed with the WT receptor shows an inability to generate intracellular increases in cyclic adenosine monophosphate (cAMP)14. Karakaya et al, also located deletion of exon 3 in one woman who was a poor responder. On the other hand, women with deletion of exon 6 appeared to have an increased ovarian response5.
Deletion of exon 2 and 5 has been located in the mouse FSHR15 and deletion of exons 4,5 and 9 in the cattle16. These species however, show a different normal ovarian function with multiple dominant follicles developing in each cycle compared to only one in the human, emphasizing the fact that variation in FSHR splicing may be necessary among species.
Research has also shown that deletion of exon 6 and 9 and insertion of intron 8 can be found in infertile men11.
This is the first study to investigate alternative splicing of the FSHR in Greek women undergoing IVF treatment. The presence of FSHR spliced variants in women undergoing IVF varied from 35% in North American5 women to 8% in Turkey14, showing great variation among populations. Researches however believe that a clinical association does exist especially in the group of women with deletion of exon 2.
In our study, alternatively spliced variants were detected in 16% of patients;three cases of deletion of exon 9 and two cases of insertion in intron 8 (Figure 2). These five women were found to have similar characteristics (BMI, basal FSH level) as well as ovarian response to exogenous gonadotrophins (assessed through E2 level on the day of hCG administration, the total number of oocytes retrieved and oocyte maturation rate). In an attempt to gather more information, pregnancy rates were also evaluated even though it was not one of our primary end points. Once again the data were inconclusive as two live births were recorded, one in a woman showing deletion of exon 9 and one with insertion of intron 8.
Figure 2. Schematic representation of FSH variants detected in the study.
Even though the data in this study are limited, it is believed that the presence of a variant FSHR may be associated with an abnormal response to FSH. All the previous published studies have a common finding, a low detection rate of FSHR splice variants. This is also the case in our study with a variant detection rate of 16%. One possible hypothesis to explain this is that alternative splicing of the FSHR may be mediated by the action of hCG administered for oocyte maturation. Screening of a greater number of subjectsis necessary in order to define the association of FSHR spliced variants with ovarian response and female infertility. Future research should specifically aim at women who show a poor response to ovarian stimulation as a connection has been made by Gerasimova et al and Karakaya et al in the past5,14. Collecting cumulus cells from poor responders however, may be challenging as a great number of these cycles are cancelled and moreover a limited number of oocytes is usually retrieved in these cycle therefore yielding lower numbers of cumulus cells. As a result, the experimental method must be tailored in such a way to optimise results even though the amount RNA is very limited. It is believed that technological advances will be able to overcome these difficulties and allow easier identification of FSHR variants in an attempt to formulate a genetic profile prior to ovulation induction. Our research is ongoing and we are confident that in the near future we will have more insight on the effect of FSHR splice variants especially in poor responders.
Conclusion
FSHR splice variants may prove to play a role in how a woman responds to ovarian stimulation. Unfortunately, the rate of detection of FSHR variants is low but it is believed that a deletion of exon 2 may be highly associated with poor response to exogenous gonadotrophins. The variants detected in this study including Greek women undergoing IVF did not show any clinical correlation to ovarian response which is in accordance to what Gerasimova et al have previously published5. Further research is necessary to investigate a larger group of subjects aiming more specifically at poor responders in Greece. This will enable us to determine whether a correlation really exists so as to utilise specific FSHR variants for optimising ovarian stimulation protocols.
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Received 30-7-2019
Revised 3-9-2019
Accepted 9-9-2019