The Effect of Follicle-Stimulating Hormone Receptor (FSHR) Polymorphism on Outcomes of Controlled Ovarian Hyperstimulation (COH) and In-vitro Fertilization and Embryo Transfer (IVF-ET)

Pharmacogenetics and Genomics

Effect of follicle-stimulating hormone receptor N680S polymorphism in the efficacy offollicle stimulating hormone stimulation on donor ovarian response.

--Manuscript Draft--

Manuscript Number: PGEN-2012-82R1

Full Title: Effect of follicle-stimulating hormone receptor N680S polymorphism in the efficacy offollicle stimulating hormone stimulation on donor ovarian response.

Article Type: Original Article

Keywords: Controlled Ovarian Hyperstimulation, COH; FSHR; SNPs; N680S.

Corresponding Author: Belen Lledo, Ph.DInstituto Bernabeu BiotechALICANTE, SPAIN

Corresponding Author SecondaryInformation:

Corresponding Author's Institution: Instituto Bernabeu Biotech

Corresponding Author's SecondaryInstitution:

First Author: Belen Lledo, Ph.D

First Author Secondary Information:

Order of Authors: Belen Lledo, Ph.D

Jaime Guerrero, MSc

Azahara Turienzo, MSc

Jose A Ortiz, PhD

Ruth Morales, PhD

Jorge Ten, PhD

Joaquin Llacer, MD

Rafael Bernabeu, PhD

Order of Authors Secondary Information:

Abstract: OBJECTIVE: Investigate whether N680S FSHR polymorphism has a predictive valuefor ovarian response to stimulation with gonadotropins and cycle outcome in our eggdonor program.METHODS: The oocyte donor candidates were selected according to InstitutoBernabeu egg donation program requirements and ASRM and ESHRE guidelines foroocyte donation. FSHR polymorphism N680S has been studied in 145 oocyte donors.All the donors underwent controlled ovarian hyperstimulation (COH) (n=355) usingurinary FSH in a GnRH antagonist protocol and receiving a GnRH agonist triggering.The main outcome measures were oocyte yield, days of stimulation, gonadotrophindosages, biochemical pregnancy, ongoing pregnancy and miscarriage rates.RESULTS: Significance differences were reported in antral follicles count (16.5 + 5.0for NN, 14.5+4.7 for NS and 14.1+3.8 for SS), number of eggs retrieved (21.5 + 9.2 forNN, 18.5 + 8.2 for NS and 19.8 + 8.9 for SS) and gonadotropin doses (2098.5 + 639.4IU for NN, 2023 +490.1 IU for NS and 2149.5 + 552.3 IU for SS) between genotypes.Clinical outcome was not affected by the N680S polymorphism on FSHR gene in eggdonors.CONCLUSIONS: In a population of fertile egg donors FSHR gene polymorphism atposition 680 is associated with different ovarian response to COH. FSHR genegenotype is an important factor for determining the prognosis of COH cycles on normoovulatory fertile women.

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Chief Editor

Pharmacogenetics and Genomics Journal

Alicante, 13 December, 2012

Dear Sir,

Please find enclosed 6 archives of the revised article entitled “Effect of follicle-

stimulating hormone receptor N680S polymorphism in the efficacy of follicle

stimulating hormone stimulation on donor ovarian response” that we are submitting

for consideration in your Journal.

The work is original, has not been published, and is not being considered for

publication elsewhere.

The work is in agreement with the Consort guidelines, and was conducted

following the principles of the Helsinki declaration.

Please, refer all further communications to Dr. Belen Lledo.

Looking forward to hear from you,

Sincerely,

Belen Lledo, PhD

Head of Molecular Biology Deparment

Instituto Bernabeu Biotech

Cover letter

The Editor

Pharmacogenetics and Genomics

Alicante, 13th December, 2012

Dear Sir,

Ref: PGEN-2012-82, entitled "Effect of follicle-stimulating hormone receptor S680N

polymorphism in the efficacy of follicle stimulating hormone stimulation on donor ovarian

response"

Please find enclosed the reviewed paper entitled Effect of follicle-stimulating hormone

receptor S680N polymorphism in the efficacy of follicle stimulating hormone stimulation on

donor ovarian response" that we submitted for consideration in your Journal.

We address and incorporate the suggested revisions and comments. We have modified

the sentences in the manuscript and included the corrections according to reviewer’s

suggestions. We rewrite some paragraph according to reviewer’s comments. Moreover,

the text was reviewed for grammatical errors.

Responding to the reviewer 1, all the suggestions were included. Specific comments:

- We included more general and clinical characteristics data of study samples

like smoking status, age, proven fertility, fertilized eggs...etc. The table had been

divided in two.

- We clarify whether patients means egg donors or recipients.

- We expand the statistical methods.

- We had preferred to show the mean values because are the main

representation in other paper. But we had prepared a figure 1 with boxplots to

show median values and distributions.

- We had adjusted in statistical test for confounder effects.

- We had included p-values in the text and tables.

- We had included the test performed in the table.

- We had included in text which genotype group is statistical different.

- Donor characteristics that were continuous variables were tested using analysis

of variance (ANOVA) to evaluate differences among the groups. Linear

regression was applied to evaluate the effect of each polymorphism on donor

ovarian stimulation parameters adjusting for age, BMI, antral follicle count

(AFC), previous fertility and smoking status as posible confounding factors, as

these have been reported to affect ovarian response. Pearson's chi-square test

was used for categorical variables.

- The table 1, 2 and 3 had been completed.

- We had revised the data and the statistical test for results in cycle outcome and

any significance had been showed between groups. We concluded that the

FSHR genotype affect the ovarian stimulation but not the embryo implantation

rate only by the fact that it is carrying a specific genotype. Differences in

number of cases of each group could explain the statistical results.

Responding to the reviewer 2, all the suggestions were included. Specific comments:

- We have modified some sentences in the manuscript and included the

corrections.

*Response to reviewers

- We rewrite some paragraph. Moreover, the text was reviewed for grammatical

errors.

- We could not resume the two tables in one because the information

concerning different populations: oocyte donors and recipients. Moreover,

more statistical information had been included.

- The heterozygotes genotypes were corrected and typed as NS only.

Responding to the reviewer 3, all the suggestions were included. Specific comments:

- The ancestry information of the study cohort is Caucasian, we included in the

population characteristics.

- We expand the statistical methods and include more statistical data like p-

value in tables and text.

This study had the approval of the Instituto Bernabeu Review Board. And we declare that

we don´t have any commercial or financial interests in this paper.

Please, refer all further communications to Belen Lledo.

Looking forward to hear from you,

Sincerely,

Dra. Belen Lledó

Molecular Biology

Instituto Bernabeu Biotech

1

TITLE: Effect of follicle-stimulating hormone receptor N680S polymorphism in the efficacy 1

of follicle stimulating hormone stimulation on donor ovarian response. 2

SHORT TITLE: Polymorphism of FSHR and ovarian response. 3

AUTHORS: Belen Lledo, PhDa, Jaime Guerrero, MScb, Azahara Turienzo, MSca, Jose 4

Antonio Ortiz, PhDa, Ruth Morales PhDa, Jorge Ten, PhDb, Joaquin Llacer, MDb and 5

Rafael Bernabeu, MD PhDa,b. 6

ADDRESSES: 7

a IB Biotech, 03016, Alicante, Spain. 8

b Instituto Bernabeu of Fertility and Gynecology, 03016, Alicante, Spain 9

10

E-MAIL CORRESPONDING AUTHOR: [email protected] 11

REPRINT REQUEST: Belén Lledó PhD, IB Biotech. 12

Avda. Albufereta, 31. 03016, Alicante, Spain 13

FAX: 0034 96 515 13 28 14

E-mail: [email protected] 15

16

Conflicts of interest and source of funding none declared. 17

18

19

*Revised manuscript with changes highlighted

2

STRUCTURED ABSTRACT 1

OBJECTIVE: Investigate whether N680S FSHR polymorphism has a predictive value for 2

ovarian response to stimulation with gonadotropins and cycle outcome in our egg 3

donor program. 4

METHODS: The oocyte donor candidates were selected according to Instituto Bernabeu 5

egg donation program requirements and ASRM and ESHRE guidelines for oocyte 6

donation. FSHR polymorphism N680S has been studied in 145 oocyte donors. All the 7

donors underwent controlled ovarian hyperstimulation (COH) (n=355) using urinary FSH 8

in a GnRH antagonist protocol and receiving a GnRH agonist triggering. The main 9

outcome measures were oocyte yield, days of stimulation, gonadotrophin dosages, 10

biochemical pregnancy, ongoing pregnancy and miscarriage rates. 11

RESULTS: Significance differences were reported in antral follicles count (16.5 + 5.0 for 12

NN, 14.5+4.7 for NS and 14.1+3.8 for SS), number of eggs retrieved (21.5 + 9.2 for NN, 18.5 13

+ 8.2 for NS and 19.8 + 8.9 for SS) and gonadotropin doses (2098.5 + 639.4 IU for NN, 2023 14

+490.1 IU for NS and 2149.5 + 552.3 IU for SS) between genotypes. Clinical outcome was 15

not affected by the N680S polymorphism on FSHR gene in egg donors. 16

CONCLUSIONS: In a population of fertile egg donors FSHR gene polymorphism at 17

position 680 is associated with different ovarian response to COH. FSHR gene genotype 18

is an important factor for determining the prognosis of COH cycles on normo ovulatory 19

fertile women. 20

21

22

KEYWORDS: Controlled Ovarian Hyperstimulation, COH; FSHR; SNPs; N680S. 23

24

3

INTRODUCTION 1

It is well recognized that an individual variability in response to drugs exists [1]. Although 2

many nongenetic factors influence the effects of medications, during recent years it 3

has become evident that genetic factors could explain the differences between 4

individuals in drug response. These differences are due to sequence variants in genes 5

encoding drug targets [2]. 6

More than 19 million single-nucleotide polymorphisms (SNPs) have been identified in the 7

human genome [3]. Some of these SNPs have already been associated with changes in 8

the effects of drugs. The challenge for pharmacogenetics is to establish the relation 9

between gene variant and medication response, to develop diagnostic tests that can 10

predict drug action and adjust therapy accordingly [2]. 11

Follicle-stimulating hormone (FSH) is a key factor in human reproduction. FSH and its 12

receptor (FSHR) play a major role in follicular development and regulation of 13

steroidogenesis in the ovary [4]. The FSHR gene is localized on chromosome 2p21 and 14

spans a region of 54kb [5]. It consists of ten exons [6]. 15

Controlled ovarian hyperstimulation (COH) using FSH alone or associated with luteinizing 16

hormone (LH), in different regimens, is a largely used strategy in Assisted Reproductive 17

Techniques (ART). Ovarian response to FSH, however, varies widely among women 18

undergoing ovarian stimulation [7]. Approximately 9%–24% of women undergoing IVF 19

respond more poorly than expected to the ovarian stimulation protocol prescribed in 20

accordance with their clinical characteristics [7]. Advance identification of patients 21

who will elicit a poor response to standard treatment would be of great clinical 22

advantages for such patients. Various predictive markers of COH outcome have been 23

proposed such as age [8], ovarian reserve [9], hormonal status [10] and cigarette 24

smoking [11]. Besides these parameters, genetic variability also seems to be an 25

important factor. Application of pharmacogenetics to ovarian response may predict 26

stimulation success [12] but also to adjust and design the doses prior the overtaken the 27

treatment. 28

4

Almost one thousand SNPs have been located in the FSHR gene, but only a few are in 1

the exons. Two of them, located at codon 307 and 680, are related to ovarian 2

response. The first is found in the extracellular domain (codon 307) and the second is in 3

the intracellular domain (codon 680). Both SNPs affect gene function by changing the 4

properties of the gene product and consequently modifying the response to FSH [13]. 5

Threonine (T) can be substituted by alanine (A) at position 307 and serine (S) can be 6

substituted by asparagine (N) at position 680. These polymorphisms are in linkage 7

disequilibrium resulting in the most frequent allelic combination T307-N680 and A307-8

S680. In order to simplify, most studies focus almost exclusively on polymorphisms at 9

codon 680. Clinical studies have demonstrated that N680S polymorphism determines 10

ovarian response to FSH stimulation in patients undergoing IVF treatment [14, 15, 16]. 11

The amount of FSH needed for COH to achieve similar peak estradiol levels was lower in 12

women with the genotype N/N at position 680, suggesting a lower sensitivity to FSH for 13

the S680 allele and a poor response to gonadotropins [17]. Patients with the S680 allele 14

need more FSH during the stimulation phase. In fact S/S genotype leads to higher FSH 15

serum levels and a prolonged cycle which suggest a lower sensitivity to exogenous FSH. 16

At the time of hCG administration, estradiol levels per oocyte retrieved for IVF in the S/S 17

group were significantly lower as compared with the levels in the N/S and N/N groups. 18

This lower response could be overcome by increasing the dose of FSH [18]. There is an 19

association between ovarian reserve and response and this could suggest an important 20

role of FSHR genotype. However, recent studies have been published reporting that 21

ovarian reserve markers is not associated with FSHR N680S polymorphism [19,20]. 22

In order to show a correlation between N680S FSHR polymorphism and COH, we 23

proposed evaluating the ovarian stimulation in a non confusion model like patients from 24

egg donation program because egg donors are young, fertile women with normal 25

ovulations and there is a minimal variability in oocyte and embryo quality. The goal of 26

this study is to investigate whether N680S FSHR polymorphism have a predictive value 27

for ovarian response to stimulation with FSH, oocyte yielded, dose of FSH, days of 28

stimulation and cycle outcome during an oocyte donor program. 29

5

MATERIALS AND METHODS 1

Study Population 2

Egg donations are the best model to evaluate the determinants of implantation for 3

several reasons. First of all, oocyte and embryo quality vary minimally, as donors are 4

young women with normal ovulation. Secondly, the preparation of the endometrium is 5

similar, as all recipients receive the same hormone replacement protocol. 6

The selection and recruitment of donors is carried out in our clinic following strict quality 7

criteria, including an extensive chromosomal and genetic evaluation. All donors were 8

caucasians and met the legal requirements in Spain (Spanish Law 14/2006). They must 9

be between 18 and 35 years of age, healthy, and with no family history of hereditary 10

diseases. The donors undergo a complete gynecological examination, karyotype and 11

screening for infectious diseases such as HIV, hepatitis B and C, gonoccocia, and 12

syphilis. In addition to the legal requirements, we perform genetic screening for cystic 13

fibrosis, fragile X and and thalasemia. Furthermore, guidelines of both ASRM and 14

ESHRE for oocyte donors are followed. 15

In this study we include the results of FSHR 680 polymorphism in 145 oocyte donors. 16

These donors performed 355 COH cycles and the results from stimulation and cycle 17

outcome were included in the present research. 18

All the subjects included in the study gave their informed consent to collect peripheral 19

blood samples suitable for molecular analysis. This study involved only retrospective 20

analysis of anonymous medical records and was approved by the Instituto Bernabeu 21

Institutional Review Board. 22

Genotyping 23

DNA was isolated from peripheral blood lymphocytes according to the manufacturer 24

instructions (Wizard® Genomic DNA Purification Kit, Promega, USA) and stored at 4ºC. 25

Analysis of FSHR gene polymorphism at position 680 was determined using the 26

predesigned TaqMan allelic discrimination assays (rs 6166, Life Tecnologies Corporation, 27

Carlsbad, NM). Real time PCR was performed using the StepOne plus system from 28

6

Applied Biosystems in accordance with the manufacturer’s instructions. Analysis was 1

performed in accordance with the instructions for the device used. 2

Ovarian stimulation and oocyte retrieval 3

After following the Spanish Fertility Act requirements, all the donors received a 4

controlled ovarian stimulation protocol with tailored doses of urinary FSH, (Fostipur, 5

Angelini. Spain). Gonadotropin stimulation started from day 2 of menstrual cycles, with 6

doses varying between 150 and 300 IU/day depending of the age of the donor, body 7

mass index and antral follicle count. The GnRH antagonist cetrorelix 0.25 mg/day 8

(Cetrotide; Merck-Serono. Paris) was introduced according to a multiple-dose, flexible 9

protocol. In all cases, triggering was exclusively performed with 0.4 mg of subcutaneous 10

triptorelin (Decapeptyl; Ipsen Pharma. Paris). Ovarian response was monitored by 11

transvaginal ultrasound and plasma estradiol concentrations. Oocytes were aspirated 12

36 hours after analogue administration by transvaginal, ultrasound-guided needle 13

aspiration under sedation. Sperm and oocyte preparation, fertilization, embryo culture 14

and transfer were performed according to IVF laboratory guidelines. 15

Recipient protocol 16

Recipient women carried out a standard protocol as previously reported [21]. In short, 17

patients with ovarian activity received in the luteal phase of the previous cycle either 18

birth control pills or analogue depot (Decapeptyl depot 3.75; Ipsen Pharma. Paris). On 19

the other hand, menopausal patients were treated with a sequential regime of 20

estrogen and progesterone the month prior the real treatment. Oral estradiol valerate 21

(Progynova, Schering) or estradiol patches releasing 50 micrograms diary (Dermestril 50; 22

Rottapharm-Madaus) was used in increasing doses for the endometrial preparation. 23

Patients received up to 6 mg estradiol valerate per day or 3 patches every other day 24

and the duration of the treatment varied in accordance with the availability of a 25

phenotypically matched donor, ranging from 14 to 24 days. After 13 days of E2 valerate 26

administration, endometrial thickness and pattern were tested. If a trilaminar pattern 27

was observed in a ≥7 mm endometrium, the aforementioned dose of E2 therapy was 28

continued at least until the pregnancy test was performed two weeks later. If the 29

7

endometrium was not seen to be sufficiently developed, doses of E2 valerate were 1

increased to 8 mg/day or 4 patches. From the day of oocyte retrieval until the 2

pregnancy test was performed, 600 mg of micronized progesterone (Utrogestan; Seid. 3

Paris) were administered vaginally daily 4

Statistical Analysis 5

Values are presented as averages ± SD for continuous data and percentages for 6

categorical variables. Data were analyzed with Statistical Package for the Social 7

Sciences (SPSS) software (version 16.0, SPSS, Inc., Chicago, IL, USA). The primary 8

endpoints were gonadotropin consumption, estimulation length and total number of 9

oocytes retrieved in donors. Donor characteristics that were continuous variables are 10

expressed as means ± SDs and were tested using analysis of variance (ANOVA) to 11

evaluate differences among the groups. Pearson's chi-square test was used for 12

categorical variables. Linear regression was applied to evaluate the effect of each 13

polymorphism on donor ovarian stimulation parameters adjusting for age, antral follicle 14

count (AFC), previous fertility and smoking status as posible confounding factors, as 15

these have been reported to affect ovarian response. A p<0.05 was considered 16

significant.17

8

RESULTS 1

FSHR N680S polymorphism genotyping 2

All female candidates for the Instituto Bernabeu egg donation program have to pass a 3

psychological evaluation and a gynecological check-up according to ASRM and 4

ESHRE guidelines for oocyte donors. After this, infectious and genetic studies are carried 5

out in order to assure the health of the offspring. Karyotype, screening for alfa and beta 6

thalassemia, cystic fibrosis, and fragile X genetic screening are part of our strict 7

selection and recruitment protocol and consequently have been performed on all of 8

our egg donor candidates. A total of 145 women were examined for the FSHR variant 9

N680S in this study. In total, the results indicated that 61 donors had SS genotype (42%), 10

58 showed NS genotype (40%) and in 26 donors NN was detected (18%). The genotype 11

frequencies were consistent with Hardy-Weinberg equilibrium. 12

Ovarian stimulation 13

The 145 oocyte donors included in this study performed 355 COH cycles. Table 1 show 14

the donor general and clinical characteristics, no differences were observed in donor 15

age (25.3+3.9y, 26.1+3.7y and 25.4+3.9y; p=0.144) and previous proven fertility (91.4%, 16

89.4%, 88.7%; p=0.784) between SS, NS and NN genotype for FSHR 680 polymorphism 17

respectively. Concerning smoking status in NN genotype group only 46.6 % smoke 18

versus 62.1 for NS and 76.2 for SS groups with a statistical significance (p<0.001). 19

Differences in antral follicle count were reported between genotypes: 16.5 + 5.0 for NN, 20

14.5+4.7 for NS and 14.1+3.8 for SS (p=0.001). Table 2 summarizes the different groups of 21

ovarian stimulation parameters in the 355 COH cycles. Various predictive markers of 22

COH outcome have been proposed such as age, ovarian reserve and cigarette 23

smoking. In order to avoid confounder effects of these predictive markers we 24

performed adjusted statistical analysis. We report significant differences in the number 25

of eggs retrieved between genotypes, patient carrier of NN genotype retrieved more 26

oocytes (21.5 + 9.2) than NS (18.5 + 8.2) and SS (19.8 + 8.9)(p<0.001). Gonadotropin 27

doses correlated with respect to the genotype in FSHR polymorphism. Women from the 28

SS group used significantly more gonadotropin (2149.5+552.3 IU) than the other groups 29

9

(2098.5+639.4 IU for NN and 2023.5.7+490.1 IU for NS; p<0.001). Days of stimulation were 1

correlated with the N680S FSHR polymorphism. The days of stimulation for women from 2

NS (11.4+1.8) and NN (11.4+1.6) were lower than SS group (11.8+1.3)(p<0.001). Figure 1 3

show the boxplots for gonadotrophin used, stimulation length and retrieved oocyte 4

among the FSHR genotype. 5

Cycle Outcome 6

Egg donation treatment outcomes are given in Table 3. We compare the results 7

between genotypes (SS, NS, NN). Overall, 355 COH cycles were considered for this 8

study and no significant differences in cycle outcome were observed between 9

genotypes (table 3). No differences were observed in recipient patient age (40.7+ 4.2 10

for SS, 41.1 +4.4 for NS and 39.9+4.8 for NN; p=0.074), endometrial thickness (8.8±1.7 for 11

SS, 8.4±1.6 for NS and 9.2±1.5 for NN; p=0.310), days of hormone replacement therapy 12

(19.2+4.2 for SS, 18.5+3.8 for NS and 18.8+4.1 for NN; p=0.420), number of oocyte 13

received (12.8+ 3.0 for SS, 12.9 +4.0 for NS and 13.4+3.6 for NN; p=0.467), fertilization rate 14

(p=0.501 for conventional IVF and p=0.706 for ICSI), fertilization technique or the day of 15

embryo transfer (data not shown). There were no significant differences with respect to 16

biochemical pregnancy (70.0% for SS, 70.2% for NS and 65.2% for NN; p=0.731), ongoing 17

pregnancy rate (56.2% for SS, 55.6% for NS and 47.0% for NN; p=0.410), miscarriage rate 18

(13.3% for SS, 8.3% for NS and 16.1% for NN; p=0.419) and implantation rate (41.6% for SS, 19

41.6% for NS and 34.1% for NN; p=0.161). 20

21

10

DISCUSSION 1

To our knowledge, these data show for the first time the relation between FSHR N680S 2

polymorphism and ovarian stimulation and clinical outcome using a non-confounding 3

model of egg donation. Our data suggests that antral follicle count and ovarian 4

stimulation is affected by the polymorphism genotype on FSHR gene. The number of 5

oocytes yielded, the days of stimulation and the gonadotropin dosage are associated 6

with the genotype in N680S polymorphism on FHSR gene. And otherwise in previous 7

studies the clinical outcome is not associated with the genotype of S680 polymorphism. 8

The genotypic variance of the FSHR was reported for the first time by Aitomäki et al in 9

1995 [22]. After that time the possibility has been considered as to whether 10

polymorphism on FSHR gene affects the ovarian response to exogenous gonadotropins. 11

Perez Moraga et al [17] studied 161 infertile women undergoing IVF and observed that 12

SS genotypes requires higher gonadotropin doses and have higher basal FSH levels 13

than other genotypes. Jun et al [23] showed a higher dose of gonadotropin for SS 14

group than others and higher oocyte retrieval and pregnancy rate compared for NN 15

group than others. Similarly, Sudo et al [24], de Castro [25] and a recent study published 16

by Sheikhha et al [26] showed the same results. Interestingly corroborating with these 17

studies, our study shows that in the SS group the gonadotropin dosage is higher and the 18

oocytes retrieved are less than other genotype groups in COH from fertile egg donors. 19

The higher gonadotropin consumption in the SS group could be explained by the fact 20

that patients with SS genotype have increased basal FSH level and tend to require 21

large FSH doses as reported in the Yao metanalysis. These findings implied that women 22

with the SS variant of the receptor were more resistant to FSH action than women 23

carrying the others variants [26, 24]. However Yao did not found association between 24

the number of oocytes and genotype. One explanation for this might be that there is a 25

reflection of the IVF procedure: the FSH dose of poor responders is raised to achieve an 26

adequate number of eggs and the dose of good responders is lowered to avoid 27

hyperstimulation. The only clinical trial on gene variants and COH outcome conducted 28

so far has confirmed the previous finding of N680S polymorphism effect, indicating that 29

11

lower FSH sensitivity of SS carriers may be overcome by higher FSH doses during COH 1

protocols [18]. 2

COH is a crucial step in ART. Successful outcomes following ART are largely dependent 3

on the patient’s response to controlled COH. And the risk of an inadequate response to 4

stimulation requiring cycle cancellation is probable. In order to improve the chances of 5

successful outcome doses should be tailored according to the patient’s characteristics. 6

There are several factors that can predict the ovarian response such as age [8], ovarian 7

reserve [9], hormonal status [10] and cigarette smoking [11]. Ovarian reserve is probably 8

the most important factor in determining success rates after IVF. Gonadotropin dosage 9

for ovarian stimulation usually increases with decreasing ovarian reserve, though it is 10

unknown whether such increases in stimulation improve oocyte yield. However, a 11

recent meta-analysis has shown that markers of ovarian reserve have only modest 12

value in predicting the response to gonadotropins [28]. From this assumption recent 13

research have been tried to show an association between FSHR genotype and ovarian 14

reserve markers such as AMH and antral follicle count [19, 20] but they did not find it. 15

This is not surprising, given that ovarian reserve consists of primordial follicles that are not 16

activated by FSH and the expression of FSHR is not relevant to these follicles [29]. 17

Concerning AMH serum levels and FSHR genotype a recent study shows that AMH 18

decreased gonadotropin stimulated aromatase expression and surprisingly AMH also 19

reduced FSHR mRNA expression [30]. Therefore, AMH inhibits factors affecting FSH 20

sensitivity [30]. Moreover Greb et al [31] observed women with SS genotype had an 21

earlier drop in the luteal secretion to the products estradiol, progesterones and inhibin 22

A, a fact that was associated with the earlier regression of the corpus luteum. As a 23

consequence of this decreased negative feedback of luteal secretion to the pituitary, 24

FSH secretion rose earlier, and this rise appeared to remain constantly higher 25

throughout the follicular phase. Surpassing the FSH threshold level earlier stimulates and 26

prolongs the FSH-dependent phase of follicular maturation: this may explain the 27

increased number of visible antral follicles in women with SS genotype according basal 28

to FSH levels. Oocyte donation is the best model to evaluate the determinants of 29

12

stimulation and embryo implantation potential. Donors are young women of similar age 1

with normal ovarian function and, in our egg donation program, with previous proven 2

fertility. Our data suggest that in our study population different antral follicle count are 3

between genotype groups. NN patients have higher antral follicle count than other 4

genotypes. Our results disagree with these previous researches. One explanation is 5

heterogeneity between infertile patients in previous studies and the homogeneity of 6

characteristics in present study mainly concerning age. 7

The question of whether genetic variation in FSHR is associated with pregnancy rates 8

remains controversial [18, 23, 32, 2] and requires further studies in large populations. Our 9

data suggest that there is no difference between genotypes, but our results come from 10

an egg donation program and the effect of genotype is not related to embryo 11

implantation. 12

The assumption that application of pharmacogenetis to the problem of searching 13

markers to measure ovarian reserve and predicting ovarian response may predict 14

treatment response is true [12]. However an individual with a clinical disorder is not the 15

product of the single gene disruption. It is embedded with the context of that 16

individual’s entire genome and environment [33]. In fact, some others genes related to 17

follicular growth could also play an important role in determining the response to OH. 18

Other factors such as polymorphism of the estrogen receptor and and CYP19 19

aromatase [25] and bone morphogenetic protein 15 (BMP15) [34] could be related to 20

response to exogenous FSH. The search for optimal biomarkers is ongoing for an 21

accurate prognosis of the ovarian response to exogenous gonadotropins [35]. 22

In conclusion, this investigation reveals that in a population of fertile egg donors FSHR 23

gene polymorphism at position 680 is associated with different ovarian response to 24

COH. FSHR gene genotype is an important factor for determining the prognosis of COH 25

cycles on fertile women with normal ovulation. Genotyping FSHR N680S together with 26

some additional markers may therefore provide a means of identifying a group of poor 27

responders before infertility treatment is initiated. 28

29

13

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Endocrinol 1994;12:265-71. 13

6. Simoni M, Gromoll J, Nieschlag E. The follicle- stimulating hormone receptor: biochemistry, 14

molecular biology, physiology, and pathophysiology. Endocr Rev 1997; 18:739–773 15

7. Oehninger S. Ovulation induction in IVF. Minerva Ginecol 2011; 63:137-56. 16

8. Kligman I, Rosenwaks Z. Differentiating clinical profiles: predicting good responders, poor 17

responders, and hyperresponders. Fertil Steril 2001; 76:1185-90. 18

9. Coccia ME, Rizzello F. Ovarian reserve. Ann N Y Acad Sci 2008; 1127:27-30. 19

10. Freour T, Masson D, Mirallie S, Jean M, Bach K, Dejoie T, Barriere P. Active smoking 20

compromises IVF outcome and affects ovarian reserve. Reprod Biomed Online 21

2008;16:96-102. 22

11. Haller K, Salumets A, Uibo R. Anti-FSH antibodies associate with poor outcome of ovarian 23

stimulation in IVF. Reprod Biomed Online 2008;16:350-5. 24

12. Fauser BC, Diedrich K, Devroey P. Predictors of ovarian response: progress towards 25

individualized treatment in ovulation induction and ovarian stimulation. Hum Reprod 26

Update 2008;14:1-14. 27

13. Simoni M, Tempfer CB, Destenaves B, Fauser BC. Functional genetic polymorphisms and 28

female reproductive disorders: Part I: Polycystic ovary syndrome and ovarian response. 29

Hum Reprod Update 2008;14:459-84. 30

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14. Altmäe S, Hovatta O, Stavreus-Evers A, Salumets A. Genetic predictors of controlled 1

ovarian hyperstimulation: where do we stand today? Hum Reprod Update 2011;17:813-2

28. 3

15. Yao Y, Ma CH, Tang HL, Hu YF. Influence of follicle-stimulating hormone receptor (FSHR) 4

Ser680Asn polymorphism on ovarian function and in-vitro fertilization outcome: a meta-5

analysis. Mol Genet Metab 2011;103:388-93. 6

16. Laan M, Grigorova M, Huhtaniemi IT. Pharmacogenetics of follicle-stimulating hormone 7

action. Curr Opin Endocrinol Diabetes Obes 2012;19:220-7. 8

17. Perez Mayorga M, Gromoll J, Behre HM, Gassner C, Nieschlag E, Simoni M. Ovarian 9

response to follicle-stimulating hormone (FSH) stimulation depends on the FSH receptor 10

genotype. J Clin Endocrinol Metab 2000; 85:3365-9. 11

18. Behre HM, Greb RR, Mempel A, Sonntag B, Kiesel L, Kaltwasser P, Seliger E, Röpke F, 12

Gromoll J, Nieschlag E et al. Significance of a common single nucleotide polymorphism in 13

exon 10 of the follicle-stimulating hormone (FSH) receptor gene for the ovarian response 14

to FSH: a pharmacogenetic approach to controlled ovarian hyperstimulation. 15

Pharmacogenet Genomics. 2005;15:451-6. 16

19. Binder H, Strick R, Zaherdoust O, Dittrich R, Hamori M, Beckmann MW, Oppelt PG. 17

Assessment of FSHR variants and antimüllerian hormone in infertility patients with a 18

reduced ovarian response to gonadotropin stimulation. Fertil Steril 2012;97:1169-1175. 19

20. Mohiyidden L, Newman WG, McBurney H, Mulugeta B, Roberts SA, Nardo LG. Follicle-20

stimulating hormone receptor gene polymorphisms are not associated with ovarian 21

reserve markers. Fertil Steril 2012;97:677-681. 22

21. Bernabeu R, Roca M, Torres A, Ten J. Indomethacin effect on implantation rates in oocyte 23

recipients. Hum Reprod. 2006;21:364-9. 24

22. Aitomäki K, Pakarinen P, Sistonen P, Tapanainen J, Gromoll J. Mutation in the follicle-25

stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian 26

failure. Cell 1995; 82:959-68. 27

23. Jun JK, Yoon JS, Ku SY, Choi YM, Hwang KR, Park SY et al. Follicle stimulating hormone 28

receptor gene polymorphism and ovarian response to controlled ovarian 29

hyperstimulation for IVF-ET. J Hum Genet 2006;51:665-70. 30

24. Sudo S, Kudo M, Wada S, Sato O, Hsueh AJ, Fujimoto S. Genetic and functional analyses 31

of polymorphisms in the human FSH receptor gene. Mol Hum Reprod 2006;8:893-9. 32

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25. De Castro F, Moron FJ, Montoro L, Galan JJ, Hernandez DP, Padilla ES, Ramires-Lorca R, 1

Real LM, Ruiz A. Human controlled ovarían hyperstimulation outcome is a polygenic trait. 2

Pharmacogenetics 2004;14:285-293. 3

26. Sheikhha MH, Eftekhar M, Kalantar SM. Investigating the association between 4

polymorphism of follicle-stimulating hormone receptor gene and ovarian response in 5

controlled ovarian hyperstimulation. J Hum Reprod Sci 2011;4:86-90. 6

27. De Castro F, Ruiz R, Montoro L, Perez-Hernandez D, Sanchez-Casas Padilla E, Real LM et 7

al. Role of follicle-stimulating hormone receptor Ser680Asn polymorphism in the efficacy 8

of follicle stimulating hormone. Fertil Steril 2003; 80:571-6. 9

28. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests 10

predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006;12:685-718. 11

29. Fortune JE, Yang MY, Muruvi W. The earliest stages of follicular development: follicle 12

formation and activation. Soc Reprod Fertil Suppl 2010;67:203-16. 13

30. Pellatt L, Rice S, Dilaver N, Heshri A, Galea R, Brincat M, Brown K, Simpson ER, Mason HD. 14

Anti-Müllerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human 15

granulosa cells. Fertil Steril 2011;96:1246-51. 16

31. Greb RR, Grieshaber K, Gromoll J, Sonntag B, Nieschlag E, Kiesel L, Simoni M. A common 17

single nucleotide polymorphism in exon 10 of the human follicle stimulating hormone 18

receptor is a major determinant of length and hormonal dynamics of the menstrual 19

cycle. J Clin Endocrinol Metab 2005;90:4866-72. 20

32. Klinkert ER, Velde ER, Weima S, van Zandvoort PM, Hanssen RG, Nilsson PR et al. FSH 21

receptor genotype is associated with pregnancy but not with ovarian response in IVF. 22

Reprod Biomed Online 2006;13:687-95. 23

33. Dipple KM, Phelan JK, McCabe ERB. Consequences of complexity within biological 24

networks: robustness and health or vulnerability and disease. Mol Genet Metab 25

2001;74:45-50. 26

34. Morón FJ, de Castro F, Royo JL, Montoro L, Mira E, Sáez ME, Real LM, González A, Mañes 27

S, Ruiz A. Bone morphogenetic protein 15 (BMP15) alleles predict over-response to 28

recombinant follicle stimulation hormone and iatrogenic ovarian hyperstimulation 29

syndrome (OHSS). Pharmacogenet Genomics 2006;16:485-95. 30

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35. Twigt JM, Hammiche F, Sinclair KD, Beckers NG, Visser JA, Lindemans J, de Jong FH, Laven 1

JS, Steegers-Theunissen RP. Preconception folic acid use modulates estradiol and 2

follicular responses to ovarian stimulation. J Clin Endocrinol Metab 2011;96:E322-9. 3

4

17

Figure 1. Boxplots for ovarian stimulation data among FSHR genotype. 1

2

1

TITLE: Effect of follicle-stimulating hormone receptor N680S polymorphism in the efficacy 1

of follicle stimulating hormone stimulation on donor ovarian response. 2

SHORT TITLE: Polymorphism of FSHR and ovarian response. 3

AUTHORS: Belen Lledo, PhDa, Jaime Guerrero, MScb, Azahara Turienzo, MSca, Jose 4

Antonio Ortiz, PhDa, Ruth Morales PhDa, Jorge Ten, PhDb, Joaquin Llacer, MDb and 5

Rafael Bernabeu, MD PhDa,b. 6

ADDRESSES: 7

a IB Biotech, 03016, Alicante, Spain. 8

b Instituto Bernabeu of Fertility and Gynecology, 03016, Alicante, Spain 9

10

E-MAIL CORRESPONDING AUTHOR: [email protected] 11

REPRINT REQUEST: Belén Lledó PhD, IB Biotech. 12

Avda. Albufereta, 31. 03016, Alicante, Spain 13

FAX: 0034 96 515 13 28 14

E-mail: [email protected] 15

16

Conflicts of interest and source of funding none declared. 17

18

19

*Revised manuscript- clean copy

2

STRUCTURED ABSTRACT 1

OBJECTIVE: Investigate whether N680S FSHR polymorphism has a predictive value for 2

ovarian response to stimulation with gonadotropins and cycle outcome in our egg 3

donor program. 4

METHODS: The oocyte donor candidates were selected according to Instituto Bernabeu 5

egg donation program requirements and ASRM and ESHRE guidelines for oocyte 6

donation. FSHR polymorphism N680S has been studied in 145 oocyte donors. All the 7

donors underwent controlled ovarian hyperstimulation (COH) (n=355) using urinary FSH 8

in a GnRH antagonist protocol and receiving a GnRH agonist triggering. The main 9

outcome measures were oocyte yield, days of stimulation, gonadotrophin dosages, 10

biochemical pregnancy, ongoing pregnancy and miscarriage rates. 11

RESULTS: Significance differences were reported in antral follicles count (16.5 + 5.0 for 12

NN, 14.5+4.7 for NS and 14.1+3.8 for SS), number of eggs retrieved (21.5 + 9.2 for NN, 18.5 13

+ 8.2 for NS and 19.8 + 8.9 for SS) and gonadotropin doses (2098.5 + 639.4 IU for NN, 2023 14

+490.1 IU for NS and 2149.5 + 552.3 IU for SS) between genotypes. Clinical outcome was 15

not affected by the N680S polymorphism on FSHR gene in egg donors. 16

CONCLUSIONS: In a population of fertile egg donors FSHR gene polymorphism at 17

position 680 is associated with different ovarian response to COH. FSHR gene genotype 18

is an important factor for determining the prognosis of COH cycles on normo ovulatory 19

fertile women. 20

21

22

KEYWORDS: Controlled Ovarian Hyperstimulation, COH; FSHR; SNPs; N680S. 23

24

3

INTRODUCTION 1

It is well recognized that an individual variability in response to drugs exists [1]. Although 2

many nongenetic factors influence the effects of medications, during recent years it 3

has become evident that genetic factors could explain the differences between 4

individuals in drug response. These differences are due to sequence variants in genes 5

encoding drug targets [2]. 6

More than 19 million single-nucleotide polymorphisms (SNPs) have been identified in the 7

human genome [3]. Some of these SNPs have already been associated with changes in 8

the effects of drugs. The challenge for pharmacogenetics is to establish the relation 9

between gene variant and medication response, to develop diagnostic tests that can 10

predict drug action and adjust therapy accordingly [2]. 11

Follicle-stimulating hormone (FSH) is a key factor in human reproduction. FSH and its 12

receptor (FSHR) play a major role in follicular development and regulation of 13

steroidogenesis in the ovary [4]. The FSHR gene is localized on chromosome 2p21 and 14

spans a region of 54kb [5]. It consists of ten exons [6]. 15

Controlled ovarian hyperstimulation (COH) using FSH alone or associated with luteinizing 16

hormone (LH), in different regimens, is a largely used strategy in Assisted Reproductive 17

Techniques (ART). Ovarian response to FSH, however, varies widely among women 18

undergoing ovarian stimulation [7]. Approximately 9%–24% of women undergoing IVF 19

respond more poorly than expected to the ovarian stimulation protocol prescribed in 20

accordance with their clinical characteristics [7]. Advance identification of patients 21

who will elicit a poor response to standard treatment would be of great clinical 22

advantages for such patients. Various predictive markers of COH outcome have been 23

proposed such as age [8], ovarian reserve [9], hormonal status [10] and cigarette 24

smoking [11]. Besides these parameters, genetic variability also seems to be an 25

important factor. Application of pharmacogenetics to ovarian response may predict 26

stimulation success [12] but also to adjust and design the doses prior the overtaken the 27

treatment. 28

4

Almost one thousand SNPs have been located in the FSHR gene, but only a few are in 1

the exons. Two of them, located at codon 307 and 680, are related to ovarian 2

response. The first is found in the extracellular domain (codon 307) and the second is in 3

the intracellular domain (codon 680). Both SNPs affect gene function by changing the 4

properties of the gene product and consequently modifying the response to FSH [13]. 5

Threonine (T) can be substituted by alanine (A) at position 307 and serine (S) can be 6

substituted by asparagine (N) at position 680. These polymorphisms are in linkage 7

disequilibrium resulting in the most frequent allelic combination T307-N680 and A307-8

S680. In order to simplify, most studies focus almost exclusively on polymorphisms at 9

codon 680. Clinical studies have demonstrated that N680S polymorphism determines 10

ovarian response to FSH stimulation in patients undergoing IVF treatment [14, 15, 16]. 11

The amount of FSH needed for COH to achieve similar peak estradiol levels was lower in 12

women with the genotype N/N at position 680, suggesting a lower sensitivity to FSH for 13

the S680 allele and a poor response to gonadotropins [17]. Patients with the S680 allele 14

need more FSH during the stimulation phase. In fact S/S genotype leads to higher FSH 15

serum levels and a prolonged cycle which suggest a lower sensitivity to exogenous FSH. 16

At the time of hCG administration, estradiol levels per oocyte retrieved for IVF in the S/S 17

group were significantly lower as compared with the levels in the N/S and N/N groups. 18

This lower response could be overcome by increasing the dose of FSH [18]. There is an 19

association between ovarian reserve and response and this could suggest an important 20

role of FSHR genotype. However, recent studies have been published reporting that 21

ovarian reserve markers is not associated with FSHR N680S polymorphism [19,20]. 22

In order to show a correlation between N680S FSHR polymorphism and COH, we 23

proposed evaluating the ovarian stimulation in a non confusion model like patients from 24

egg donation program because egg donors are young, fertile women with normal 25

ovulations and there is a minimal variability in oocyte and embryo quality. The goal of 26

this study is to investigate whether N680S FSHR polymorphism have a predictive value 27

for ovarian response to stimulation with FSH, oocyte yielded, dose of FSH, days of 28

stimulation and cycle outcome during an oocyte donor program. 29

5

MATERIALS AND METHODS 1

Study Population 2

Egg donations are the best model to evaluate the determinants of implantation for 3

several reasons. First of all, oocyte and embryo quality vary minimally, as donors are 4

young women with normal ovulation. Secondly, the preparation of the endometrium is 5

similar, as all recipients receive the same hormone replacement protocol. 6

The selection and recruitment of donors is carried out in our clinic following strict quality 7

criteria, including an extensive chromosomal and genetic evaluation. All donors were 8

caucasians and met the legal requirements in Spain (Spanish Law 14/2006). They must 9

be between 18 and 35 years of age, healthy, and with no family history of hereditary 10

diseases. The donors undergo a complete gynecological examination, karyotype and 11

screening for infectious diseases such as HIV, hepatitis B and C, gonoccocia, and 12

syphilis. In addition to the legal requirements, we perform genetic screening for cystic 13

fibrosis, fragile X and and thalasemia. Furthermore, guidelines of both ASRM and 14

ESHRE for oocyte donors are followed. 15

In this study we include the results of FSHR 680 polymorphism in 145 oocyte donors. 16

These donors performed 355 COH cycles and the results from stimulation and cycle 17

outcome were included in the present research. 18

All the subjects included in the study gave their informed consent to collect peripheral 19

blood samples suitable for molecular analysis. This study involved only retrospective 20

analysis of anonymous medical records and was approved by the Instituto Bernabeu 21

Institutional Review Board. 22

Genotyping 23

DNA was isolated from peripheral blood lymphocytes according to the manufacturer 24

instructions (Wizard® Genomic DNA Purification Kit, Promega, USA) and stored at 4ºC. 25

Analysis of FSHR gene polymorphism at position 680 was determined using the 26

predesigned TaqMan allelic discrimination assays (rs 6166, Life Tecnologies Corporation, 27

Carlsbad, NM). Real time PCR was performed using the StepOne plus system from 28

6

Applied Biosystems in accordance with the manufacturer’s instructions. Analysis was 1

performed in accordance with the instructions for the device used. 2

Ovarian stimulation and oocyte retrieval 3

After following the Spanish Fertility Act requirements, all the donors received a 4

controlled ovarian stimulation protocol with tailored doses of urinary FSH, (Fostipur, 5

Angelini. Spain). Gonadotropin stimulation started from day 2 of menstrual cycles, with 6

doses varying between 150 and 300 IU/day depending of the age of the donor, body 7

mass index and antral follicle count. The GnRH antagonist cetrorelix 0.25 mg/day 8

(Cetrotide; Merck-Serono. Paris) was introduced according to a multiple-dose, flexible 9

protocol. In all cases, triggering was exclusively performed with 0.4 mg of subcutaneous 10

triptorelin (Decapeptyl; Ipsen Pharma. Paris). Ovarian response was monitored by 11

transvaginal ultrasound and plasma estradiol concentrations. Oocytes were aspirated 12

36 hours after analogue administration by transvaginal, ultrasound-guided needle 13

aspiration under sedation. Sperm and oocyte preparation, fertilization, embryo culture 14

and transfer were performed according to IVF laboratory guidelines. 15

Recipient protocol 16

Recipient women carried out a standard protocol as previously reported [21]. In short, 17

patients with ovarian activity received in the luteal phase of the previous cycle either 18

birth control pills or analogue depot (Decapeptyl depot 3.75; Ipsen Pharma. Paris). On 19

the other hand, menopausal patients were treated with a sequential regime of 20

estrogen and progesterone the month prior the real treatment. Oral estradiol valerate 21

(Progynova, Schering) or estradiol patches releasing 50 micrograms diary (Dermestril 50; 22

Rottapharm-Madaus) was used in increasing doses for the endometrial preparation. 23

Patients received up to 6 mg estradiol valerate per day or 3 patches every other day 24

and the duration of the treatment varied in accordance with the availability of a 25

phenotypically matched donor, ranging from 14 to 24 days. After 13 days of E2 valerate 26

administration, endometrial thickness and pattern were tested. If a trilaminar pattern 27

was observed in a ≥7 mm endometrium, the aforementioned dose of E2 therapy was 28

continued at least until the pregnancy test was performed two weeks later. If the 29

7

endometrium was not seen to be sufficiently developed, doses of E2 valerate were 1

increased to 8 mg/day or 4 patches. From the day of oocyte retrieval until the 2

pregnancy test was performed, 600 mg of micronized progesterone (Utrogestan; Seid. 3

Paris) were administered vaginally daily 4

Statistical Analysis 5

Values are presented as averages ± SD for continuous data and percentages for 6

categorical variables. Data were analyzed with Statistical Package for the Social 7

Sciences (SPSS) software (version 16.0, SPSS, Inc., Chicago, IL, USA). The primary 8

endpoints were gonadotropin consumption, estimulation length and total number of 9

oocytes retrieved in donors. Donor characteristics that were continuous variables are 10

expressed as means ± SDs and were tested using analysis of variance (ANOVA) to 11

evaluate differences among the groups. Pearson's chi-square test was used for 12

categorical variables. Linear regression was applied to evaluate the effect of each 13

polymorphism on donor ovarian stimulation parameters adjusting for age, antral follicle 14

count (AFC), previous fertility and smoking status as posible confounding factors, as 15

these have been reported to affect ovarian response. A p<0.05 was considered 16

significant.17

8

RESULTS 1

FSHR N680S polymorphism genotyping 2

All female candidates for the Instituto Bernabeu egg donation program have to pass a 3

psychological evaluation and a gynecological check-up according to ASRM and 4

ESHRE guidelines for oocyte donors. After this, infectious and genetic studies are carried 5

out in order to assure the health of the offspring. Karyotype, screening for alfa and beta 6

thalassemia, cystic fibrosis, and fragile X genetic screening are part of our strict 7

selection and recruitment protocol and consequently have been performed on all of 8

our egg donor candidates. A total of 145 women were examined for the FSHR variant 9

N680S in this study. In total, the results indicated that 61 donors had SS genotype (42%), 10

58 showed NS genotype (40%) and in 26 donors NN was detected (18%). The genotype 11

frequencies were consistent with Hardy-Weinberg equilibrium. 12

Ovarian stimulation 13

The 145 oocyte donors included in this study performed 355 COH cycles. Table 1 show 14

the donor general and clinical characteristics, no differences were observed in donor 15

age (25.3+3.9y, 26.1+3.7y and 25.4+3.9y; p=0.144) and previous proven fertility (91.4%, 16

89.4%, 88.7%; p=0.784) between SS, NS and NN genotype for FSHR 680 polymorphism 17

respectively. Concerning smoking status in NN genotype group only 46.6 % smoke 18

versus 62.1 for NS and 76.2 for SS groups with a statistical significance (p<0.001). 19

Differences in antral follicle count were reported between genotypes: 16.5 + 5.0 for NN, 20

14.5+4.7 for NS and 14.1+3.8 for SS (p=0.001). Table 2 summarizes the different groups of 21

ovarian stimulation parameters in the 355 COH cycles. Various predictive markers of 22

COH outcome have been proposed such as age, ovarian reserve and cigarette 23

smoking. In order to avoid confounder effects of these predictive markers we 24

performed adjusted statistical analysis. We report significant differences in the number 25

of eggs retrieved between genotypes, patient carrier of NN genotype retrieved more 26

oocytes (21.5 + 9.2) than NS (18.5 + 8.2) and SS (19.8 + 8.9)(p<0.001). Gonadotropin 27

doses correlated with respect to the genotype in FSHR polymorphism. Women from the 28

SS group used significantly more gonadotropin (2149.5+552.3 IU) than the other groups 29

9

(2098.5+639.4 IU for NN and 2023.5.7+490.1 IU for NS; p<0.001). Days of stimulation were 1

correlated with the N680S FSHR polymorphism. The days of stimulation for women from 2

NS (11.4+1.8) and NN (11.4+1.6) were lower than SS group (11.8+1.3)(p<0.001). Figure 1 3

show the boxplots for gonadotrophin used, stimulation length and retrieved oocyte 4

among the FSHR genotype. 5

Cycle Outcome 6

Egg donation treatment outcomes are given in Table 3. We compare the results 7

between genotypes (SS, NS, NN). Overall, 355 COH cycles were considered for this 8

study and no significant differences in cycle outcome were observed between 9

genotypes (table 3). No differences were observed in recipient patient age (40.7+ 4.2 10

for SS, 41.1 +4.4 for NS and 39.9+4.8 for NN; p=0.074), endometrial thickness (8.8±1.7 for 11

SS, 8.4±1.6 for NS and 9.2±1.5 for NN; p=0.310), days of hormone replacement therapy 12

(19.2+4.2 for SS, 18.5+3.8 for NS and 18.8+4.1 for NN; p=0.420), number of oocyte 13

received (12.8+ 3.0 for SS, 12.9 +4.0 for NS and 13.4+3.6 for NN; p=0.467), fertilization rate 14

(p=0.501 for conventional IVF and p=0.706 for ICSI), fertilization technique or the day of 15

embryo transfer (data not shown). There were no significant differences with respect to 16

biochemical pregnancy (70.0% for SS, 70.2% for NS and 65.2% for NN; p=0.731), ongoing 17

pregnancy rate (56.2% for SS, 55.6% for NS and 47.0% for NN; p=0.410), miscarriage rate 18

(13.3% for SS, 8.3% for NS and 16.1% for NN; p=0.419) and implantation rate (41.6% for SS, 19

41.6% for NS and 34.1% for NN; p=0.161). 20

21

10

DISCUSSION 1

To our knowledge, these data show for the first time the relation between FSHR N680S 2

polymorphism and ovarian stimulation and clinical outcome using a non-confounding 3

model of egg donation. Our data suggests that antral follicle count and ovarian 4

stimulation is affected by the polymorphism genotype on FSHR gene. The number of 5

oocytes yielded, the days of stimulation and the gonadotropin dosage are associated 6

with the genotype in N680S polymorphism on FHSR gene. And otherwise in previous 7

studies the clinical outcome is not associated with the genotype of S680 polymorphism. 8

The genotypic variance of the FSHR was reported for the first time by Aitomäki et al in 9

1995 [22]. After that time the possibility has been considered as to whether 10

polymorphism on FSHR gene affects the ovarian response to exogenous gonadotropins. 11

Perez Moraga et al [17] studied 161 infertile women undergoing IVF and observed that 12

SS genotypes requires higher gonadotropin doses and have higher basal FSH levels 13

than other genotypes. Jun et al [23] showed a higher dose of gonadotropin for SS 14

group than others and higher oocyte retrieval and pregnancy rate compared for NN 15

group than others. Similarly, Sudo et al [24], de Castro [25] and a recent study published 16

by Sheikhha et al [26] showed the same results. Interestingly corroborating with these 17

studies, our study shows that in the SS group the gonadotropin dosage is higher and the 18

oocytes retrieved are less than other genotype groups in COH from fertile egg donors. 19

The higher gonadotropin consumption in the SS group could be explained by the fact 20

that patients with SS genotype have increased basal FSH level and tend to require 21

large FSH doses as reported in the Yao metanalysis. These findings implied that women 22

with the SS variant of the receptor were more resistant to FSH action than women 23

carrying the others variants [26, 24]. However Yao did not found association between 24

the number of oocytes and genotype. One explanation for this might be that there is a 25

reflection of the IVF procedure: the FSH dose of poor responders is raised to achieve an 26

adequate number of eggs and the dose of good responders is lowered to avoid 27

hyperstimulation. The only clinical trial on gene variants and COH outcome conducted 28

so far has confirmed the previous finding of N680S polymorphism effect, indicating that 29

11

lower FSH sensitivity of SS carriers may be overcome by higher FSH doses during COH 1

protocols [18]. 2

COH is a crucial step in ART. Successful outcomes following ART are largely dependent 3

on the patient’s response to controlled COH. And the risk of an inadequate response to 4

stimulation requiring cycle cancellation is probable. In order to improve the chances of 5

successful outcome doses should be tailored according to the patient’s characteristics. 6

There are several factors that can predict the ovarian response such as age [8], ovarian 7

reserve [9], hormonal status [10] and cigarette smoking [11]. Ovarian reserve is probably 8

the most important factor in determining success rates after IVF. Gonadotropin dosage 9

for ovarian stimulation usually increases with decreasing ovarian reserve, though it is 10

unknown whether such increases in stimulation improve oocyte yield. However, a 11

recent meta-analysis has shown that markers of ovarian reserve have only modest 12

value in predicting the response to gonadotropins [28]. From this assumption recent 13

research have been tried to show an association between FSHR genotype and ovarian 14

reserve markers such as AMH and antral follicle count [19, 20] but they did not find it. 15

This is not surprising, given that ovarian reserve consists of primordial follicles that are not 16

activated by FSH and the expression of FSHR is not relevant to these follicles [29]. 17

Concerning AMH serum levels and FSHR genotype a recent study shows that AMH 18

decreased gonadotropin stimulated aromatase expression and surprisingly AMH also 19

reduced FSHR mRNA expression [30]. Therefore, AMH inhibits factors affecting FSH 20

sensitivity [30]. Moreover Greb et al [31] observed women with SS genotype had an 21

earlier drop in the luteal secretion to the products estradiol, progesterones and inhibin 22

A, a fact that was associated with the earlier regression of the corpus luteum. As a 23

consequence of this decreased negative feedback of luteal secretion to the pituitary, 24

FSH secretion rose earlier, and this rise appeared to remain constantly higher 25

throughout the follicular phase. Surpassing the FSH threshold level earlier stimulates and 26

prolongs the FSH-dependent phase of follicular maturation: this may explain the 27

increased number of visible antral follicles in women with SS genotype according basal 28

to FSH levels. Oocyte donation is the best model to evaluate the determinants of 29

12

stimulation and embryo implantation potential. Donors are young women of similar age 1

with normal ovarian function and, in our egg donation program, with previous proven 2

fertility. Our data suggest that in our study population different antral follicle count are 3

between genotype groups. NN patients have higher antral follicle count than other 4

genotypes. Our results disagree with these previous researches. One explanation is 5

heterogeneity between infertile patients in previous studies and the homogeneity of 6

characteristics in present study mainly concerning age. 7

The question of whether genetic variation in FSHR is associated with pregnancy rates 8

remains controversial [18, 23, 32, 2] and requires further studies in large populations. Our 9

data suggest that there is no difference between genotypes, but our results come from 10

an egg donation program and the effect of genotype is not related to embryo 11

implantation. 12

The assumption that application of pharmacogenetis to the problem of searching 13

markers to measure ovarian reserve and predicting ovarian response may predict 14

treatment response is true [12]. However an individual with a clinical disorder is not the 15

product of the single gene disruption. It is embedded with the context of that 16

individual’s entire genome and environment [33]. In fact, some others genes related to 17

follicular growth could also play an important role in determining the response to OH. 18

Other factors such as polymorphism of the estrogen receptor and and CYP19 19

aromatase [25] and bone morphogenetic protein 15 (BMP15) [34] could be related to 20

response to exogenous FSH. The search for optimal biomarkers is ongoing for an 21

accurate prognosis of the ovarian response to exogenous gonadotropins [35]. 22

In conclusion, this investigation reveals that in a population of fertile egg donors FSHR 23

gene polymorphism at position 680 is associated with different ovarian response to 24

COH. FSHR gene genotype is an important factor for determining the prognosis of COH 25

cycles on fertile women with normal ovulation. Genotyping FSHR N680S together with 26

some additional markers may therefore provide a means of identifying a group of poor 27

responders before infertility treatment is initiated. 28

29

13

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4

17

Figure 1. Boxplots for ovarian stimulation data among FSHR genotype. 1

2

FigureClick here to download high resolution image

Table 1. Donor baseline characteristics in relation to FSHR S680 genotype.

AFC: Antral follicle count

Test performed for statistical analysis ANOVA for donor age and AFC.

Test performed for statistical analysis chi-square for proven fertility and smoker.

FSHR S680 genotype SS (61) NS (58) NN (26) P

Donor Age (years) 25.3±3.9 26.1±3.7 25.4±3.9 0.144

Proven fertility (%) 91.4 89.4 88.7 0.784

Smoker (%) 76.2 62.1 46.6 <0.001

AFC 14.1±3.8 14.5±4.7 16.5±5.0 0.001

Table

Table 2. Donor ovarian stimulation data in relation to FSHR S680 genotype.

Test performed for statistical analysis regression linear using SS genotype as reference

FSHR S680 genotype SS (151) NS (142) NN (62) P

(unadjusted)

P

(adjusted)

P

(age-adjusted)

P

(fertility-adjusted)

P

(smoker-adjusted)

P

(AFC-adjusted)

Stimulation length (days) 11.8±1.3 11.4±1.8 11.4±1.6 0.026 <0.001 0.007 0.048 0.008 0.035

Gonadotropin used (IU) 2149.5±552.3 2023.5±490.1 2098.5±639.4 0.142 <0.001 <0.001 0.268 0.021 0.005

No. of retrieved oocytes 19.8±8.9 18.5±8.2 21.5±9.2 0.062 <0.001 0.098 0.001 0.322 0.008

Table

Table 3. Recipients characteristics and cycle outcomes according to egg donor genotype.

FSHR S680 genotype SS (151) NS (142) NN (62) P

Recipient age (years) 40.7±4.2 41.4±4.4 39.9±4.8 0.074

Follicular phase length (days) 19.1±4.2 18.5±3.9 18.8±4.2 0.420

Recipient endometrial

thickness (mm) 8.8±1.7 8.4±1.6 9.2±1.5 0.310

No. of oocytes received 12.8±3.0 12.9±4.0 13.4±3.6 0.467

2PN conventional IVF 7.0±4.1 7.8±3.6 7.3±3.9 0.501

2 PN ICSI 8.3±2.8 8.4±3.8 8.0±3.4 0.706

Transferred embryos 1.8±0.4 1.9±0.4 1.9±0.5 0.217

Positive β-HCG (%) 70.0 70.2 65.2 0.731

Clinical pregnancy rate (%) 56.2 55.6 47.0 0.410

Implantation rate (%) 41.6 41.6 34.1 0.161

Miscarriage rate (%) 13.3 8,3 16.1 0.419

Test performed for statistical analysis ANOVA

Table

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