|Year : 2019 | Volume
| Issue : 2 | Page : 85-91
Genetics of female infertility: Molecular study of newborn ovary homeobox gene in poor ovarian responders
Osamah Batiha1, Nour Alhoda Alahmad1, Amer Sindiani2, Khaldon Bodoor1, Sherin Shaaban1, Mohammad Al-Smadi3
1 Department of Applied Biological Sciences, Jordan University of Science and Technology, Irbid, Jordan
2 Department of Obstetrics and Gynecology, Jordan University of Science and Technology, Irbid, Jordan
3 Department of Reproductive Endocrinology and IVF Unit, King Hussein Medical Center, Amman, Jordan
|Date of Web Publication||17-Jun-2019|
Dr. Osamah Batiha
Department of Applied Biological Sciences, Jordan University of Science and Technology, Irbid
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Newborn ovary homeobox (NOBOX) gene plays a critical role in the transcriptional regulation of oocyte-specific genes. Previous studies have demonstrated a pathogenic effect of NOBOX variants on premature ovarian insufficiency (POI) patients. Poor ovarian response (POR) is a risk factor for POI. Therefore, genetic variants in the NOBOX gene may also be studied as risk factors for POR development. Aims: The aim of the study is to investigate the association between seven known NOBOX single-nucleotide polymorphisms (SNPs) and POR in Jordanian females. Settings and Design: This was a case–control study of 60 females with POR for controlled ovarian hyperstimulation and 59 healthy females with no history of reproductive problems. Blood samples were collected from the participants and seven SNPs of NOBOX gene were screened. Subjects and Methods: DNA was extracted from blood samples. Polymerase chain reaction with primers specific for seven known SNPs in NOBOX gene was used to amplify the specified region within the gene followed by Sanger sequencing. Results: The seven SNPs investigated in this study, namely, rs77587352 (c.271G>T, p. Gly91Trp), rs7800847 (c.349C>T, p. Arg117Trp), rs193303102 (c.907C>T, p. Arg303X), rs193303103 (c.1025G>C, p. Ser342Thr), rs193303104 (c.1048G>T, p. Val350Leu), rs201947677 (c.1064G>A, p. Arg355His), and rs146227301 (c.1856C>T, p. Pro619Leu), only represent the wild-type allele in both females with POR and healthy participants. Conclusions: The results show that only monomorphic genotype of the NOBOX variants was found in Jordanian females studied.
Keywords: Female infertility, newborn ovary homeobox gene, poor ovarian response
|How to cite this article:|
Batiha O, Alahmad NA, Sindiani A, Bodoor K, Shaaban S, Al-Smadi M. Genetics of female infertility: Molecular study of newborn ovary homeobox gene in poor ovarian responders. J Hum Reprod Sci 2019;12:85-91
|How to cite this URL:|
Batiha O, Alahmad NA, Sindiani A, Bodoor K, Shaaban S, Al-Smadi M. Genetics of female infertility: Molecular study of newborn ovary homeobox gene in poor ovarian responders. J Hum Reprod Sci [serial online] 2019 [cited 2019 Sep 17];12:85-91. Available from: http://www.jhrsonline.org/text.asp?2019/12/2/85/260493
| Introduction|| |
Ovarian reserve describes the female reproductive potential in terms of both quality and quantity of oocytes., Decreased or diminished ovarian reserve represents the premature loss of oocytes and poor oocyte development, a common condition occurs naturally after the age of 40 leading to menopause., Younger females with decreased ovarian response are usually referred to reproductive clinics and undergo a controlled ovarian hyperstimulation (COH) protocol to obtain mature oocytes. In some cases, they achieve a poor response to COH manifested by low number of retrieved oocytes or complete absence of oocytes. Females with such condition are diagnosed with poor ovarian responders (POR). More than one-third of females undergoing assisted reproductive technology (ART) is diagnosed with POR.
The prevalence of POR in different countries ranges between 9% and 24%. In 2007, the American Society for Reproductive Medicine and the Society for ART reported that at least 50% of canceled in vitro fertilization (IVF) cycles were due to POR.
The etiology of POR is unknown; however, age, advanced endometriosis, ovarian surgery, pelvic adhesion, smoking, and high body mass index are considered to be risk factors associated with low ovarian reserve.
Furthermore, POR is linked to mutations in genes involved in oocyte production. For example, several studies identified an association between genetic polymorphisms in the gonadotropin hormone genes and their receptors and POR.
Newborn ovary homeobox gene (NOBOX) is an oocyte-specific homeobox gene which is expressed in mice primordial and growing oocytes and is essential for mice folliculogenesis. Several studies point to an essential role for NOBOX in oocyte development such as the lack of the gene is linked to the loss of postnatal oocyte, blocks the transition from primordial to growing follicles, and downregulates oocyte preferential genes such as Oct4 and Gdf-9.
NOBOX gene is located on chromosome 7q35 and is composed of 10 exons.
NOBOX transcript in humans is present in the ovary, testis, and pancreas. NOBOX expression in the oocyte was detected from the primordial stage of the ovarian follicle until the MII stage of mature oocyte. Microarray analysis of newborn mouse ovaries lacking NOBOX gene showed that 28 oocyte-specific genes were downregulated more than five-fold, whereas only five oocyte-specific genes were upregulated. Some of these affected genes have specific roles in signaling pathways related to oocyte developments such as actin binding (Ttid gene), aldehyde dehydrogenase (E330034G19Rik gene), aldehyde reductase (Aldrl6 gene), arginine deiminase (Padi6 gene), exocytosis (Rims1 gene), histone/oocyte specific (H1foo gene), microtubule movement (Dnahc8 gene), notch signaling pathway (Jag1 gene), olfactory receptor (Olfr976 gene), oligoadenylate synthetase (Oas1e, Oas1d, Oas1c, and Oas1h genes), protein kinase (Mos and 2610028F08Rik genes), Ring finger protein (Rfpl4 gene), secreted factor (Fetub, Astl, Gdf9, and Oosp1 genes), Solute carrier (Slc6a20 gene), and transcription (Pou5f1, 1700008J08Rik, and Sall4). Others they do not seem to have a clear function (C86187, D5Ertd577e, BC052883, Nlrp4f, Nlrp14, Oog1, BG071013, D9Ertd414e, BM229829, D11Ertd636e, AK005675, E130009J12Rik, and Nlrp4c genes). The results of these studies confirm the critical role of the NOBOX gene in oocyte gene regulation and oogenesis. In addition, it implies that mutations in NOBOX may lead to POR and infertility.
The association between NOBOX and premature ovarian insufficiency (POI) disorder has been studied in several populations [Table 1].,,,,, POI is a condition of amenorrhea, estrogen deficiency, and menopausal follicle-stimulating hormone (FSH) levels in young women (<40).
|Table 1: Newborn ovary homeobox gene variations in premature ovarian insufficiency in different populations|
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Poor ovarian response (POR) to gonadotropin is an indicator of ovarian failure, whereas poor responders have a higher chance of being diagnosed with POI.,,,
In this study, we investigated the frequency of seven known NOBOX single-nucleotide polymorphisms (SNPs) [Table 2] in females with POR. The selected SNPs have been previously associated with POI disease in different populations.,,,
|Table 2: A summary of the seven studied single-nucleotide polymorphisms in this study|
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| Subjects and Methods|| |
Selection and description of participants
This case-controlled prospective study included 152 POR patients. Blood samples were collected from different IVF centers in Jordan (King Hussein medical city, Istishari hospital, Prince Rashid Hospital, Islamic hospital, and Al-Amal Maternity Hospital) in the period of 2014–2017.
Sixty participants suffering from POR were included with the age range of 20–46 years according to the European Society of Human Reproduction and Embryology criteria. Patients with at least two of the following standards were included [Supplementary Table]: (1) anti-Müllerian hormone (AMH) level <1.1 ng/ml, (2) FSH level more than 10 mIU/ml and E2 level 25–75 pg/ml on day 3 of a normal menstrual cycle, (3) the antral follicle count (AFC) is <9, and (4) the number of retrieved meiosis II oocytes is <5. Patients >40 years were included in this study only when they were diagnosed with POR before the age of 40 and were still trying ART at the time of sample selection. Ninety-one samples were excluded due to incomplete data or due to known infertility causes such as pelvic surgery, ovarian cysts, radiation therapy, and chemotherapy. Despite the long duration of sample collection, sample size was limited because of the scarcity of the disease in Jordan and the stringency of the selection criteria.
Control group included 59 whole blood samples collected from normal fertile females with age range of 24–39 years whom were able to carry out a normal pregnancy without the need to undergo any ART, and they have no medical history of pelvic surgeries, ovarian cysts, radiation therapy, and chemotherapy.
Control samples were collected from different private clinics in Amman, (King Hussein medical city and King Abdullah university hospital). The Institutional Review Board at Jordan University of Science and Technology and King Abdullah University Hospital (KAUH) approved the study, and informed written consent was obtained from all patients and controls.
DNA extraction and sequencing
Genomic DNA was extracted from whole blood samples using Puregene Blood Core Kit B (Qiagen) according to the manufacturer's instructions.
Seven SNPs have been studied in NOBOX gene covered by three different pairs of primers. All primers were designed through SnapGene software using the NOBOX gene sequence obtained from the Ensembl genome browser according to the transcript ID number (ENST00000467773.1). The primers were synthesized at Princess Haya Biotechnology Centre, Irbid, Jordan. Primers sequences, products size, included polymorphisms, and their cycling conditions are listed in [Table 3].
|Table 3: Primer sequences used for polymerase chain reaction in the study and their cycling conditions|
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The polymerase chain reaction (PCR) products were loaded in 2% agarose gel in 1X TBE buffer at 120V for 45 min to determine the product size. 50 bp DNA ladder was used to determine the band sizes, and the gel was stained with ethidium bromide then displayed by GelDoc-It 310 imaging system (UVP, USA). PCR products were purified using ExoSAP-IT PCR Product Cleanup kit (Affymetrix) and then loaded in the ABI 310 Genetic Analyzer (ABI Prism310, Applied Biosystems) at Princess Haya Biotechnology Centre. ChromasPro software (Technelysium Pty Ltd) was used to analyze the sequencing data.
| Results|| |
Seven genetic variations in the NOBOX gene in 60 POR females with an average age of 33.6 + 6.4 years (20–46) were compared to 59 healthy fertile females under the age of 40 (24–39). [Table 4] summarizes the selection categories and the numbers of participants investigated.
The average AMH level of the cases involved was 0.344 + 0.257 ng/ml (0.03–0.98), FSH level was 19.55 + 13.7 mIU/ml (10.6–62.6), whereas the average AFC number was 3.52 + 1.64 (0–6) and 2.25 + 1.27 (0–4) for the average number of meiosis II oocytes retrieved after stimulation. According to the sequencing analysis, there were no differences between cases and controls in all seven SNPs, all of them had the wild-type alleles. [Table 5] summarizes the results of the studied SNPs in both cases and controls. In general, environmental endocrine disruptors, tobacco, genetic mutations, endometriomas, ovarian surgery, chemotherapy, and short menstrual cycles are factors that affect the stimulation process in assisted reproduction cycles. In this study, we excluded samples with a history of endometriomas, ovarian surgeries, and chemotherapy.
|Table 5: Summary of sequencing results of the common newborn ovary homeobox single-nucleotide polymorphisms among all samples|
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| Discussion|| |
The process of oogenesis is tightly regulated by a set of genes, which guarantee the proper production of competent oocytes ready for fertilization. Among these genes are the NOBOX, which has been found to play important roles in oogenesis and folliculogenesis., In this work, we studied the prevalence of seven SNPs in the NOBOX gene and found that both cases (POR patients) and control groups represented the wild-type allele in all investigated SNPs; therefore, we exclude the role of NOBOX in POR in the studied Jordanian cohort.
Several previous studies have reported the association between NOBOX polymorphisms and ovarian failure syndromes such as primary ovarian insufficiency.,,, This association is not surprising as NOBOX is known as an important early regulator of both folliculogenesis and oogenesis processes. However, the relationship between NOBOX and POR has not been investigated till now. Nevertheless, several studies have shown that poor response can be an early sign of ovarian failure;,,, therefore, we determined to study the role of NOBOX in females with poor response to COH.
Several polymorphisms in the NOBOX gene have been associated with POI in populations of the USA, France, Tunisia, and Han China.,,, In a Tunisian study, mutations were found in 6.4% in patients and none of them were found in control group. In France, 5.6% of the patients with POI displayed heterozygous NOBOX mutations. Another study in France found a NOBOX loss-of-function mutation in 6.2% of POI cases.
However, in other studies in Japan and China, they found no association between NOBOX SNPs and POI., The discrepancy between these studies could be due to differences in ethnicity, selection criteria, differences in sample size, and the analysis of different regions of the NOBOX gene.
Despite the importance of the evaluation of the ovarian reserve in determining the outcome of the assisted reproductive procedures, still few indicators/biomarkers are being used. Physiological markers such as high FSH and low AMH are commonly used as indicators of the ovarian reserve., Hormones such as FSH, LH, and their receptors are important for folliculogenesis; therefore, the association between SNPs in these genes and poor response has been studied., One of these polymorphisms in the promoter of FSHB gene (rs10835638; c.-211G>T) has been associated with lower FSH level and late or longer menopause. FSH receptor is important for the FSH action, and some genetic polymorphisms have been associated with ovarian response., Finally, genetic variants in the luteinizing hormone biological function subunit β have been associated with poor response to hormonal stimulation during ART procedures which qualifies this gene as a good indicator of ovarian response.,
Lifestyle such as smoking and taking oral contraceptive pill also has directly effects on ovarian reserve and hormone levels regardless of genetic associations.
There are some limitations of this study. First, it included a small sample size which may prevent polymorphisms detection. Second, the samples were collected from different IVF centers which may cause variations in the results of hormone levels. In future studies, we recommend to collect the samples from one IVF center and to investigate the roles of other genes, in addition to incorporate females responded normally to COH as controls.
Poor response is considered a big challenge for gynecologists as well as patients to achieve successful pregnancy and to overcome infertility problems; therefore, more studies should be followed to understand the pathophysiological etiologies' behind this condition. Molecular investigation for variants in genes important in folliculogenesis and ovulation will help in optimizing better treatment of POR cases and help to improve pregnancy outcomes.
To our knowledge, this is the first study in Jordan studying the genetic causes of POR. We can exclude the probability of NOBOX variations pathogenicity for POR until proven otherwise.
| Conclusions|| |
This is the first study exploring NOBOX gene variations in poor ovarian responders. We did not find any link between POR and seven previously studied SNPs in NOBOX gene (rs77587352, rs7800847, rs193303102, rs193303103, rs193303104, rs201947677, and rs146227301) in a subset of Jordanian females. We recommend investigating the role of other genes in the future.
We would like to thank all patients and volunteers who participated in this study. This research is funded by a grant from the deanship of research at Jordan University of Science and Technology (grant # 20150244). We would also like to thank Dr. Khaldoun Al-Sherif for his cooperation in sample collection.
Financial support and sponsorship
This research is funded by a grant from the deanship of research at Jordan University of Science and Technology (grant # 20150244).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Skinner MK. Regulation of primordial follicle assembly and development. Hum Reprod Update 2005;11:461-71.
Jirge PR, Chougule SM, Gavali VG, Bhomkar DA. Impact of dehydroepiandrosterone on clinical outcome in poor responders: A pilot study in women undergoing in vitro
fertilization, using Bologna criteria. J Hum Reprod Sci 2014;7:175-80.
] [Full text]
Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro
fertilization: The Bologna criteria. Hum Reprod 2011;26:1616-24.
Oehninger S. Poor responders in in vitro
fertilization (IVF) therapy: The challenge continues. Facts Views Vis Obgyn 2011;3:101-8.
Venetis CA, Kolibianakis EM, Tarlatzi TB, Tarlatzis BC. Evidence-based management of poor ovarian response. Ann N Y Acad Sci 2010;1205:199-206.
Society for Assisted Reproductive Technology; American Society for Reproductive Medicine. Assisted reproductive technology in the United States: 2001 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2007;87:1253-66.
Badawy A, Wageah A, El Gharib M, Osman EE. Prediction and diagnosis of poor ovarian response: The dilemma. J Reprod Infertil 2011;12:241-8.
Riccetti L, De Pascali F, Gilioli L, Santi D, Brigante G, Simoni M, et al.
Genetics of gonadotropins and their receptors as markers of ovarian reserve and response in controlled ovarian stimulation. Best Pract Res Clin Obstet Gynaecol 2017;44:15-25.
Suzumori N, Yan C, Matzuk MM, Rajkovic A. Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes. Mech Dev 2002;111:137-41.
Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science 2004;305:1157-9.
National Center for Biotechnology Information, NOBOX Oogenesis Homeobox Homo sapiens (Human). Available from: https://www.ncbi.nlm.nih.gov/gene
. [Last updated on 2018 Dec 08].
Huntriss J, Hinkins M, Picton HM. CDNA cloning and expression of the human NOBOX gene in oocytes and ovarian follicles. Mol Hum Reprod 2006;12:283-9.
Choi Y, Qin Y, Berger MF, Ballow DJ, Bulyk ML, Rajkovic A. Microarray analyses of newborn mouse ovaries lacking nobox. Biol Reprod 2007;77:312-9.
Bouilly J, Roucher-Boulez F, Gompel A, Bry-Gauillard H, Azibi K, Beldjord C, et al.
New NOBOX mutations identified in a large cohort of women with primary ovarian insufficiency decrease KIT-L expression. J Clin Endocrinol Metab 2015;100:994-1001.
Bouali N, Francou B, Bouligand J, Lakhal B, Malek I, Kammoun M, et al.
NOBOX is a strong autosomal candidate gene in tunisian patients with primary ovarian insufficiency. Clin Genet 2016;89:608-13.
Bouilly J, Bachelot A, Broutin I, Touraine P, Binart N. Novel NOBOX loss-of-function mutations account for 6.2% of cases in a large primary ovarian insufficiency cohort. Hum Mutat 2011;32:1108-13.
Li L, Wang B, Zhang W, Chen B, Luo M, Wang J, et al.
Ahomozygous NOBOX truncating variant causes defective transcriptional activation and leads to primary ovarian insufficiency. Hum Reprod 2017;32:248-55.
Qin Y, Shi Y, Zhao Y, Carson SA, Simpson JL, Chen ZJ. Mutation analysis of NOBOX homeodomain in chinese women with premature ovarian failure. Fertil Steril 2009;91:1507-9.
Zhao XX, Suzumori N, Yamaguchi M, Suzumori K. Mutational analysis of the homeobox region of the human NOBOX gene in Japanese women who exhibit premature ovarian failure. Fertil Steril 2005;83:1843-4.
Hubayter ZR, Popat V, Vanderhoof VH, Ndubizu O, Johnson D, Mao E, et al.
Aprospective evaluation of antral follicle function in women with 46, XX spontaneous primary ovarian insufficiency. Fertil Steril 2010;94:1769-74.
Farhi J, Homburg R, Ferber A, Orvieto R, Ben Rafael Z. Non-response to ovarian stimulation in normogonadotrophic, normogonadal women: A clinical sign of impending onset of ovarian failure pre-empting the rise in basal follicle stimulating hormone levels. Hum Reprod 1997;12:241-3.
Nikolaou D, Lavery S, Turner C, Margara R, Trew G. Is there a link between an extremely poor response to ovarian hyperstimulation and early ovarian failure? Hum Reprod 2002;17:1106-11.
Lawson R, El-Toukhy T, Kassab A, Taylor A, Braude P, Parsons J, et al.
Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: A life table analysis. Hum Reprod 2003;18:527-33.
Szmidt NA, Bhattacharya S, Maheshwari A. Does poor ovarian response to gonadotrophins predict early menopause? A retrospective cohort study with minimum of 10-year follow-up. Hum Fertil (Camb) 2016;19:212-9.
Amaral ME, Ejzenberg D, Wajman DS, Monteleone PA, Serafini P, Soares JM Jr., et al.
Risk factors for inadequate response to ovarian stimulation in assisted reproduction cycles: Systematic review. J Assist Reprod Genet 2019;36:19-28.
Belli M, Cimadomo D, Merico V, Redi CA, Garagna S, Zuccotti M. The NOBOX protein becomes undetectable in developmentally competent antral and ovulated oocytes. Int J Dev Biol 2013;57:35-9.
Qin Y, Choi Y, Zhao H, Simpson JL, Chen ZJ, Rajkovic A. NOBOX homeobox mutation causes premature ovarian failure. Am J Hum Genet 2007;81:576-81.
Greene AD, Patounakis G, Segars JH. Genetic associations with diminished ovarian reserve: A systematic review of the literature. J Assist Reprod Genet 2014;31:935-46.
Ruth KS, Beaumont RN, Tyrrell J, Jones SE, Tuke MA, Yaghootkar H, et al.
Genetic evidence that lower circulating FSH levels lengthen menstrual cycle, increase age at menopause and impact female reproductive health. Hum Reprod 2016;31:473-81.
Gromoll J, Simoni M, Nordhoff V, Behre HM, De Geyter C, Nieschlag E. Functional and clinical consequences of mutations in the FSH receptor. Mol Cell Endocrinol 1996;125:177-82.
Casarini L, Moriondo V, Marino M, Adversi F, Capodanno F, Grisolia C, et al.
FSHR polymorphism p.N680S mediates different responses to FSH in vitro
. Mol Cell Endocrinol 2014;393:83-91.
Alviggi C, Clarizia R, Pettersson K, Mollo A, Humaidan P, Strina I, et al.
Suboptimal response to gnRHa long protocol is associated with a common LH polymorphism. Reprod Biomed Online 2011;22 Suppl 1:S67-72.
Alviggi C, Pettersson K, Longobardi S, Andersen CY, Conforti A, De Rosa P, et al.
Acommon 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.
Schuh-Huerta SM, Johnson NA, Rosen MP, Sternfeld B, Cedars MI, Reijo Pera RA. Genetic variants and environmental factors associated with hormonal markers of ovarian reserve in Caucasian and African American women. Hum Reprod 2012;27:594-608.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]