Variant-beta luteinizing hormone is not associated with poor ovarian response to controlled ovarian hyperstimulation
© Hanevik et al.; licensee BioMed Central Ltd. 2014
Received: 30 October 2013
Accepted: 12 February 2014
Published: 13 March 2014
The most common genetic variant of luteinizing hormone (LH), variant-betaLH, has a different bioactivity than the wildtype. Carrying the variant allele was associated with an increased consumption of exogenous gonadotropin to achieve optimal ovarian response for in vitro fertilization procedures (IVF). The aim of this study was to examine if variant-betaLH was also more common in patients with a poor ovarian response to exogenous gonadotropin which negatively influenced treatment outcome.
36 patients with poor ovarian response to ovarian stimulation for IVF and 98 controls with a normal response were genotyped for variant-betaLH using DNA sequencing. The carrier frequency in the control group was 17%. No association was found between poor ovarian response and variant-betaLH.
Testing patients for variant-betaLH prior to IVF is unlikely to predict poor ovarian response.
KeywordsLuteinizing hormone Genetic variant Variant-beta LH Ovarian response IVF
The most common form of genetically determined variation in luteinizing hormone (LH) structure is variant-betaLH (v-betaLH) . v-betaLH is caused by a doublet of single nucleotide polymorphisms (SNPs) in the LHB gene that induces a substitution of amino acids (LHB Trp8Arg and LHB Ile15Thr) in the beta subunit of LH . The LHB 15Thr introduces an additional glycosylation site to v-betaLH, probably affecting the serum half-life of LH and thereby its bioactivity [3–5]. Various physiological and clinical implications of v-betaLH were proposed, including infertility and premature ovarian failure [6, 7]. In the field of in vitro fertilization (IVF), patients who required an increased amount of recombinant follicular stimulating hormone (rFSH) to achieve an optimal ovarian response in controlled ovarian hyperstimulation (COH) were classified as hypo-responders . Alviggi and co-workers reported that v-betaLH was more common in hypo-responders in a series of 60 Italian IVF patients . This finding was recently confirmed in a larger series of Danish IVF patients , and is of interest when searching for genetic predictors of ovarian response to COH . Poor ovarian response (POR) has similarities to hypo-response, but is more adverse for the patient’s treatment outcome . Patients with POR have a markedly decreased chance of pregnancy from IVF treatment , whereas hypo-responders by definition achieve an optimal ovarian response . The aim of the present study was to investigate if v-betaLH was more common in IVF patients with POR. If so, this would strengthen the case raised by Alviggi and co-workers for determining v-betaLH status in patients prior to COH .
Inclusion- and exclusion criteria
Poor ovarian response
IU of rFSH per day
No. of oocytes obtained by oocyte retrieval
IU of rFSH per day
< 150 or > 200
<150 or > 200
> 40 years
> 40 years
Polycystic ovary syndrome
As for poor ovarian response.
Previous adnexal surgery
Present or previous signs of moderate or severe ovarian hyperstimulation syndrome
Ovarian endometriosis or other ovarian tumour
Ovarian cysts > 3 cm
Genotyping was done by DNA sequencing. Genomic DNA was isolated from peripheral blood according to standard procedures using EZ1 BioRobot (QIAGEN) and stored at −20°C. Each PCR-reaction contained 50 ng of genomic DNA, 0.20 M of each primer, 1x AccuPrime PCR buffer II (Invitrogen), 0.60 U AccuPrime Taq DNA polymerase (Invitrogen) and milliQ H2O to a final volume of 12.5 l. Primer sequences used: LHB F 5′-GGG TGA AGC AGT GTC CTT GT-3′ and LHB R 5′-GAA GAG GAG GCC TGA GAG TT-3′ . Cycling conditions were: 95C for 5 min, then 35 cycles of 95C for 30 s, 65C for 30 s and 72C for 30 s, then 72C for 10 min, then 4C ∞. The primers amplified most of exon 2 and 3 of LHB and gave a specific PCR-product excluding the homologous CGB genes (verified by gel electrophoresis and blast of sequence). The PCR-products were sequenced in both directions using BigDye Terminator kit v.3.1 (Applied Biosystems) and an ABI3130xl Genetic Analyzer (Applied Biosystems) according to the manufacturer’s procedures. The sequences were aligned to LHB RefSeq NG_011464.1  using SeqScape v.2.6 software (Applied Biosystems). Samples were analysed in the order they arrived at the lab, often giving samples from both groups of patients in each run. Repeated sequencing of a random 16% subset yielded 100% identical sequences.
Sample size was limited to 36 cases and 98 controls by the study-design. Chi-square statistics were used for comparison of carrier and allele frequency of v-betaLH. Prior data indicated that the carrier frequency among controls was 0.11 . If the true carrier frequency among cases was threefold, 0.33, then the null hypothesis that the carrier frequency for cases and controls were equal would be rejected with probability (power) 0.82 . Other comparisons between groups were by student’s t-test or chi-square. For all comparisons differences between groups were considered statistically significant if reaching a p-value of <0.05.
Clinical and genetic data
Poor ovarian response (n = 36)
Controls (n = 98)
Age mean (95% CI)
BMI (kg/m 2 ) mean (95% CI)
No. of patients of other ethnicities than Caucasian (%)
First time COH (%)
Early follicular phase s-FSH a mean (95% CI)
First dose of rFSH (IU) mean (95% CI)
Duration of rFSH medication (Days) mean (95% CI)
Total dose of rFSH (IU) mean (95% CI)
Oocytes retrieved mean (95% CI)
No. of embryos transferred mean (95% CI)
Live births (% of started COH)
No. of patients with v-betaLH (%)
No. of alleles with v-betaLH (%)
The present data are, to our knowledge, the first to estimate the prevalence of v-betaLH in a Norwegian population. A carrier frequency of 17% amongst normal responders to COH in an IVF population is comparable to 11% in a similar Danish IVF population . In unselected populations a wide variation of v-betaLH carrier frequency was reported ranging from 42% in Finnish Lapps through 19% in Swedes to 7% in Hispanics in the USA . No association was found between POR and v-betaLH in this study. This finding is in accordance with the only reported genome wide association study concerning ovarian response to COH . Alviggi and co-workers  suggested that IVF patients with v-betaLH lack sufficient LH activity to adequately support FSH activity in multiple follicular development, leading to hypo-response to COH as defined above. The present results suggest that the decreased ovarian response in POR patients may require a different explanation, and point to the question of whether ovarian response to COH declines gradually from normal via hypo to poor, or if the three should be considered separate situations altogether . The hypothesis proposed by Alviggi and co-workers , that carriers of v-betaLH could benefit from exogenous LH in their COH, seems unlikely to apply to POR patients from the present results; however, a different study design is required to test this properly.
Differences in numbers of oocytes retrieved, embryos transferred and live births were as expected from the inclusion- and exclusion criteria. There were no differences between groups regarding the FSH receptor SNPs reported to influence ovarian response to COH (data not shown). The control group was also not matched to the POR group, leading to differences in age and BMI between groups. Albeit small, these differences are a possible confounder in the study inasmuch as BMI was reported to influence ovarian response to COH  and the risk of POR increases with age . The use of rFSH was similar between groups, reflecting that POR was unexpected by clinicians for the patients in the POR-group.
All COHs in this study, as in the studies by Alviggi and co-workers [8, 9], were by GnRH agonist mid luteal phase down regulation. Whether v-betaLH is associated with hypo-response or POR in an antagonist protocol is unknown. However, the low endogenous level of LH in agonist cycles could arguably make v-betaLH more influential in agonist than antagonist cycles.
Genotype data from the same patients regarding other signaling systems of importance in ovarian physiology and their association with ovarian response to COH were presented earlier [13–18]. This raises the question of multiple testing in genetic association studies. However, as the present study showed negative results, concerns over false positive findings were unwarranted.
The present study had two main limitations; sample size and inclusion criteria. The above sample size calculations show that the study had adequate power to find a difference between groups if v-betaLH prevalence was three times higher in the POR group than in controls. A study with more patients could have detected smaller differences between groups. However, to avoid a high rate of false negatives when applying v-betaLH as a predictor of POR, a high prevalence of v-betaLH in the POR group is required.
The inclusion criteria for POR patients did not comply with ESHRE’s Bologna criteria . The criteria for POR found in Table 1 were set to identify patients in which POR came unexpectedly, as it is in these patients that novel predictors of ovarian response could be most useful. Also the criteria in Table 1 assured that the patients included had few other known factors that could influence their ovarian response to COH apart from the putative genetic ones such as v-betaLH. The Bologna criteria on the other hand have ‘advanced maternal age’ and ‘previous POR’, as two of three criteria for POR, making them inadequate for identification of patients with an unexpected poor response, at least retrospectively as done in this study.
Other limitations in the present study were unavailability of s-LH measurements prior to and during COH, and no data on s-AMH or antral follicle count to predict ovarian response to COH.
In conclusion this study found no association between v-betaLH and unexpected POR. This indicates that it is unlikely that testing IVF patients for v-betaLH prior to COH would contribute to predicting POR.
Body mass index
Controlled ovarian hyperstimulation
European society for human reproduction and endocrinology
Follicular stimulating hormone
Human chorionic gonadotropin
In vitro fertilization
Beta subunit of luteinizing hormone
Gene encoding beta subunit of luteinizing hormone
Polymerase chain reaction
Poor ovarian response
Recombinant follicular stimulating hormone
- v-beta LH:
Variant beta luteinizing hormone.
This research was financially supported by Telemark Hospital and the South-Eastern Norway Regional Health Authority.
- Nilsson C, Pettersson K, Millar RP, Coerver KA, Matzuk MM, Huhtaniemi IT: Worldwide frequency of a common genetic variant of luteinizing hormone: an international collaborative research. International Collaborative Research Group. Fertil Steril. 1997, 67: 998-1004. 10.1016/S0015-0282(97)81430-6.View ArticlePubMed
- Furui K, Suganuma N, Tsukahara S, Asada Y, Kikkawa F, Tanaka M, Ozawa T, Tomoda Y: Identification of 2 point mutations in the gene coding luteinizing-hormone (LH) beta-subunit, associated with immunologically anomalous LH variants. J Clin Endocrinol Metab. 1994, 78: 107-113.PubMed
- Haavisto AM, Pettersson K, Bergendahl M, Virkamaki A, Huhtaniemi I: Occurence and biological properties of a common genetic variant of luteinizing-hormone. J Clin Endocrinol Metab. 1995, 80: 1257-1263.PubMed
- Wide L, Eriksson K, Sluss PM, Hall JE: The common genetic variant of luteinizing hormone has a longer serum half-life than the wild type in heterozygous women. J Clin Endocrinol Metab. 2010, 95: 383-389. 10.1210/jc.2009-1679.PubMed CentralView ArticlePubMed
- Suganuma N, Furui K, Kikkawa F, Tomoda Y, Furuhashi M: Effects of the mutations (Trp8- > Arg and Ile15- > Thr) in human luteinizing hormone (LH) beta-subunit on LH bioactivity in vitro and in vivo. Endocrinology. 1996, 137: 831-838.PubMed
- Takahashi K, Ozaki T, Okada M, Kurioka H, Kanasaki H, Miyazaki K: Increased prevalence of luteinizing hormone beta-subunit variant in patients with premature ovarian failure. Fertil Steril. 1999, 71: 96-101. 10.1016/S0015-0282(98)00409-9.View ArticlePubMed
- Takahashi K, Kurioka H, Ozaki T, Kanasaki H, Kohsaka M, Miyazaki K, Karino K: Increased prevalence of luteinizing hormone beta-subunit variant in Japanese infertility patients. Hum Reprod. 1998, 13: 3338-3344. 10.1093/humrep/13.12.3338.View ArticlePubMed
- Alviggi C, Pettersson K, Longobardi S, Andersen CY, Conforti A, De Rosa P, Clarizia R, Strina I, Mollo A, De Placido G, Humaidan P: A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology. Reprod Biol Endocrinol. 2013, 11: 51-10.1186/1477-7827-11-51.PubMed CentralView ArticlePubMed
- Alviggi C, Clarizia R, Pettersson K, Mollo A, Humaidan P, Strina I, Coppola M, Ranieri A, D’Uva M, De Placido G: Suboptimal response to GnRHa long protocol is associated with a common LH polymorphism. Reprod Biomed Online. 2009, 18: 9-14. 10.1016/S1472-6483(10)60418-X.View ArticlePubMed
- Altmae S, Hovatta O, Stavreus-Evers A, Salumets A: Genetic predictors of controlled ovarian hyperstimulation: where do we stand today?. Hum Reprod Update. 2011, 17: 813-828. 10.1093/humupd/dmr034.View ArticlePubMed
- Ferraretti AP, La Marca A, Fauser BCJM, Tarlatzis B, Nargund G, Gianaroli L, Res EWGPO: ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011, 26: 1616-1624. 10.1093/humrep/der092.View ArticlePubMed
- Saldeen P, Kallen K, Sundstrom P: The probability of successful IVF outcome after poor ovarian response. Acta Obstet Gynecol Scand. 2007, 86: 457-461. 10.1080/00016340701194948.View ArticlePubMed
- Hanevik HI, Hilmarsen HT, Skjelbred CF, Tanbo T, Kahn JA: Single nucleotide polymorphisms in the anti-Mullerian hormone signalling pathway do not determine high or low response to ovarian stimulation. Reprod Biomed Online. 2010, 21: 616-623. 10.1016/j.rbmo.2010.05.021.View ArticlePubMed
- Hanevik HI, Hilmarsen HT, Skjelbred CF, Tanbo T, Kahn JA: A single nucleotide polymorphism in BMP15 is associated with high response to ovarian stimulation. Reprod Biomed Online. 2011, 23: 97-104. 10.1016/j.rbmo.2011.02.015.View ArticlePubMed
- Hanevik HI, Hilmarsen HT, Skjelbred CF, Tanbo T, Kahn JA: Increased risk of ovarian hyperstimulation syndrome following controlled ovarian hyperstimulation in patients with vascular endothelial growth factor + 405 cc genotype. Gynecol Endocrinol. 2012, 28: 845-849. 10.3109/09513590.2012.683056.View ArticlePubMed
- Hanevik HI, Hilmarsen HT, Skjelbred CF, Tanbo T, Kahn JA: Erratum: single nucleotide polymorphisms in the anti-Mullerian hormone signalling pathway do not determine high or low response to ovarian stimulation (vol 21, pg 613, 2010). Reprod Biomed Online. 2013, 26: 628-628. 10.1016/j.rbmo.2013.02.001.View Article
- Hanevik HI, Hilmarsen HT, Skjelbred CF, Tanbo T, Kahn JA: Erratum: a single nucleotide polymorphism in BMP15 is associated with high response to ovarian stimulation (vol 23, pg 97, 2011). Reprod Biomed Online. 2013, 26: 627-627. 10.1016/j.rbmo.2013.02.002.View Article
- Hanevik HI, Hilmarsen HT, Skjelbred CF, Tanbo T, Kahn JA: Corrigendum to Gynecological Endocrinology, 2012; 28(11): 845–849. Gynecol Endocrinol. 2013, doi:10.3109/09513590.09512013.09853035
- NCBI Reference Sequence. [http://www.ncbi.nlm.nih.gov/nuccore/NG_011464.1]
- Dupont WD, Plummer WD: Power and sample size calculations. A review and computer program. Control Clin Trials. 1990, 11: 116-128. 10.1016/0197-2456(90)90005-M.View ArticlePubMed
- van Disseldorp J, Franke L, Eijkemans R, Broekmans F, Macklon N, Wijmenga C, Fauser B: Genome-wide analysis shows no genomic predictors of ovarian response to stimulation by exogenous FSH for IVF. Reprod Biomed Online. 2011, 22: 382-388. 10.1016/j.rbmo.2010.12.006.View ArticlePubMed
- Fauser BC, Diedrich K, Devroey P: Predictors of ovarian response: progress towards individualized treatment in ovulation induction and ovarian stimulation. Hum Reprod Update. 2008, 14: 1-14.View ArticlePubMed
- Fedorcsak P, Dale PO, Storeng R, Ertzeid G, Bjercke S, Oldereid N, Omland AK, Abyholm T, Tanbo T: Impact of overweight and underweight on assisted reproduction treatment. Hum Reprod. 2004, 19: 2523-2528. 10.1093/humrep/deh485.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.