Abstract
The haptoglobin (Hp) protein has been implicated in various aspects of reproduction. One possible mechanism is through its effect on angiogenesis. Angiogenesis plays a major role in follicle production. The Hp insertion polymorphism results in the production of Hp proteins denoted Hp 1-1, 2-1, and 2-2, with markedly different angiogenic activities. We sought to determine if the number of oocytes aspirated during in vitro fertilization is related to the Hp type and to compare clinical data and treatment outcomes. We conducted a prospective non-interventional study in an academic in vitro fertilization center serving northern Israel. All patients undergoing in vitro fertilization who agreed to have their haptoglobin phenotype and clinical data evaluated anonymously were included. The main outcome measure was the number of oocytes harvested from each aspiration. The groups did not differ regarding ethnicity or BMI, though women with the Hp1-1 phenotype had a longer duration of infertility (p = 0.037) and a higher gonadotropin requirement (p = 0.024) to achieve the same treatment outcome. Women with mechanical factor infertility were more likely (p = 0.042) to have the Hp 1-1/2-1 phenotypes than the Hp2-2 phenotype. There were no differences in the number of oocytes aspirated or the pregnancy rate. In summary, we could not establish a correlation between Hp phenotype and oocyte number or IVF outcomes though the Hp2-2 phenotype may be protective against mechanical factor infertility. Further studies with a larger sample size, particularly concerning the Hp1-1 phenotype, are required in order to extend these results.
Introduction
In vitro fertilization is indicated for mechanical factor infertility, male factor infertility, and infertility refractory to other treatments. Success positively correlated with the number of oocytes aspirated and embryo quality [van Loendersloot et al. Citation2010]. Ovarian response to gonadotropins is correlated with age but varies widely [Sun et al. Citation2008].
Haptoglobin is a protein produced by liver hepatocytes and found abundantly in the serum. It has been implicated in reproductive function through its effect on inflammation, the immune system, and angiogenesis making it a protein of great interest to reproductive biologists [Bottini et al. Citation1999; Ueda et al. Citation2001].
Haptoglobin binds hemoglobin (Hb) released from blood cells after hemolysis and prevents its filtration through the kidney and secretion in the urine. The Hp protein consists of two α and two β chains bound by disulfide bonds [Levy et al. Citation2010]. Two common alleles exist at the Hp locus denoted 1 and 2. The Hp 2 allele appears to have arisen from the Hp 1 allele by an intragenic duplication event and has spread under strong genetic pressure to become the more frequent allele worldwide [Langlois and Delanghe Citation1996]. The distribution of the Hp1 allele in the Israeli Jewish and Arab populations is similar with an expected phenotype frequency for Hp1-1, Hp2-1, and Hp2-2 of 9%, 42%, and 49%, respectively [Carter and Worwood Citation2007].
Studies have shown differential protection of the different Hp phenotypes against hemoglobin's oxidative damage, whereby Hp1-1 is the most efficient phenotype. The Hp 2-2 phenotype has been associated with resistance to a number of pathogens. It is postulated that in man's transition from a hunter/gatherer to tribal society, the burden of infection increased leading to the spread of the Hp2 allele. The higher oxidative stress among Hp2-2 individuals is believed to be associated with a higher risk for degenerative diseases of modernity such as atherosclerosis [Simpson et al. Citation2011] while the decreased anti-inflammatory properties of the Hp1-1 phenotype may be associated with increased susceptibility towards malignancy [Bicho et al. Citation2009; Speeckaert et al. Citation2012].
The Hp proteins have markedly different angiogenic properties [Cid et al. Citation1993]. Ovarian function requires continual remodeling of the blood vessels to accommodate the cyclical changes which transpire during follicle and corpus luteum formation and demise [Robinson et al. Citation2009]. Several angiogenic factors have been demonstrated in the follicular fluid aspirated from women undergoing in vitro fertilization (IVF) and their concentrations were found to be a function of follicle volume [Nishigaki et al. Citation2011]. Haptoglobin α and β peptides as well as Hp proteins have been identified in the follicular fluid aspirated during IVF [Angelucci et al. Citation2006; Schweigert et al. Citation2006]. Haptoglobin alpha chains in follicular fluid, was positively correlated with a good ovarian response [Estes et al. Citation2009] and may be associated with oocyte quality [Porta et al. Citation1999]. Whereas angiogenesis is an essential stage for follicular development, Hp has been detected in the fluid of aspirated follicles, further investigation on the relationship between the Hp polymorphism and follicular development is warranted.
Results
Between April 4, 2011 and March 6, 2012, 160 patients were asked to participate and 149 consented. Two patients were withdrawn for technical reasons: the first, because not enough blood was collected; the second, because the blood failed to separate to sera after centrifugation. A total of 147 samples and IVF cycles were analyzed.
Of the 147 patients, 13 (8.8%) patients had the Hp1-1 phenotype, 66 (44.9%) had the Hp2-1 phenotype, and 68 (46.3%) had the Hp2-2 phenotype. This was in accordance with the expected frequency; and our goal of recruiting at least 63 women from the Hp2-1 and Hp2-2 phenotypes was reached.
Demographic and clinical data for the three groups are presented in . The three groups did not differ statistically regarding age, body mass index (BMI), infertility duration, primary versus secondary infertility, average follicle stimulating hormone (FSH), ethnicity, or frequency of polycystic ovary syndrome (PCOS), endometriosis, partner with male infertility as defined by World Health Organization (WHO) criteria, or unexplained infertility. There was an indication that there may be more mechanical factor infertility in the Hp2-1 group compared to the Hp2-2 group, but this did not reach the accepted threshold of statistical significance (p = 0.053). In fact when the frequency of mechanical factor infertility for groups Hp2-1 and Hp1-1 are combined and compared to Hp2-2, Hp2-2 is shown to be protective for mechanical factor infertility (p = 0.042).
Table 1. Demographic and clinical data.
Treatment results are presented in . Treatment protocols did not differ between the groups, nor did estradiol levels before the ovulation trigger, or the rate of utilizing gonadotropin releasing hormone (GnRH) agonist for the ovulation trigger. The number of oocytes aspirated and the pregnancy rate per aspiration and per embryo transfer was the same.
Table 2. Treatment and results.
The Hp1-1 group is the smallest since this phenotype is uncommon in Israel but the data shows that this group of women, compared to the other two groups, may be of an older age: 35.0 vs. 32.3 years, but this did not reach statistical significance (p = 0.097), had a longer duration of infertility 4.66 vs. 3.14 years (p = 0.037), and had greater gonadotropin requirements 3,110 vs. 2,316 units (p = 0.024) to achieve a similar number of oocytes 5.69 vs. 8.12 (p = 0.197). In other words, for every oocyte aspirated in the Hp1-1 group 546 units of gonadotropins had to be administered, as opposed to 285 units of gonadotropins for the other two groups. Pregnancy rates did not differ statistically for Hp 1-1 as compared to the other groups.
Discussion
In this study we have failed to find a difference between the Hp phenotypes in modulating the outcome of gonadotropin stimulation. There was no difference in the background and clinical parameters between the two groups other than a higher frequency of mechanical factor infertility for women with the Hp2-1 and Hp1-1 phenotype as compared to women with the Hp2-2 phenotype. Since the etiology for mechanical infertility is generally infectious pelvic disease, this provides additional evidence that the Hp2-2 phenotype may protect against infection.
Other studies have assessed the association of different Hp phenotypes and fertility. A study which assessed the Hp phenotype among white women in Italy at the time of delivery, found that the Hp1-1 phenotype was highest for the youngest women in this study group [Bottini et al. Citation1999], though it was later noted that this association was limited to smokers [Bottini et al. Citation2002]. In our study of women with infertility, women with the Hp1-1 phenotype tended to be older, have a longer duration of infertility, and require higher gonadotropin doses to reach similar results. This suggests that Hp1-1 may actually be associated with reduced fertility. Furthermore, if Hp1-1 is associated with reduced fertility, this would help explain the evolutionary penetration of the Hp2 allele in the population.
Another study which investigated the role Hp may play in in vitro fertilization treatment found a higher pregnancy rate among women with the Hp2-2 phenotype compared to the Hp2-1 phenotype (38% vs. 16%, respectively, p = 0.08) [Ueda et al. Citation2001]. The study had only one patient with the Hp1-1 phenotype from a total of fifty patients.
We could not find an association between the Hp phenotype and the number of oocytes aspirated during in vitro fertilization. The main limitation of the study presented above was our inability to recruit a sufficient number of patients with the Hp1-1 phenotype. We did find, that the Hp2 gene may be protective for mechanical factor infertility, perhaps by protecting against infection. The Hp1-1 allele is associated with older patients with a longer duration of infertility requiring higher gonadotropin doses to attain similar treatment results. Larger studies are warranted in order to ascertain the role the Hp1-1 haplotype may play in fertility and whether the Hp2-2 phenotype is indeed protective for pelvic inflammatory disease.
Materials and Methods
The study design was approved by the local ethics committee. All patients undergoing IVF therapy at our institution with gonadotrophic stimulation of ovarian follicles were candidates for inclusion. Patients were asked to participate on the day of oocyte pick up and to sign an informed consent form. Those that consented had blood drawn at the time of venflow insertion before anesthesia.
Stimulation protocols included both agonist and antagonist protocols. Oocyte retrieval was performed 33-36 h after ovulation induction with recombinant human chorionic gonadotropin (rHCG). Ovulation was triggered for high responders on the antagonist protocol with GnRH agonist. Oocyte pick up was performed under general anesthesia.
Blood was collected into tubes with a clot activator and coded to protect patient anonymity. After clot formation, tubes were centrifuged at 3,000 x revolutions per minute (RPM) for 10 min in order to separate the sera, and stored at 4oC until further handling.
Hp typing was performed on stored plasma samples by polyacrylamide gel electrophoresis. Briefly, 10 µl of Hb enriched plasma was subjected to electrophoresis in a non-denaturing gel and the gel was subsequently immersed in solution containing a congener of benzidine with a precipitate forming in the gel corresponding to the location of Hb-Hp complexes. The Hp type of the sample was determined by the banding pattern of the Hp-Hb complexes with each of the three Hp types having a characteristic banding fingerprint.
Statistical analysis
The phenotypic distribution frequency for Hp1-1, Hp2-1, and Hp2-2 was 9%, 42%, and 49%, respectively. An average of 9 oocytes per aspiration with a standard deviation of 6 based on our data prior to the initiation of this study were used. We calculated that in order to detect a difference of three oocytes per aspiration between any two groups we would need 63 patients in each group for an alpha of 0.05 and 0.8 power. We would need to recruit 150 patients to insure 63 patients from the Hp2-1 phenotype and the Hp2-2 phenotype, but since the Hp1-1 phenotype is relatively rare, we would need to recruit 700 patients in order to find enough patients with this phenotype. We therefore decided to limit the study size to that necessary to detect a difference in oocytes aspirated between patients with the Hp2-1 and Hp2-2 phenotypes.
Statistical calculations for continuous variables were done with student's t-test and for ordinal data with the Fisher's exact test.
Abbreviations
| Hp: | = | haptoglobin |
| BMI: | = | body mass index |
| AFC: | = | antral follicle count |
| GN: | = | gonadotrpin |
| rHCG: | = | recombinant human chorionic gonadotropin |
| GnRH: | = | gonadotropin releasing hormone |
| RPM: | = | revolutions per minute |
| Hb: | = | hemoglobin |
| IVF: | = | in vitro fertilization |
| PCOS: | = | polycystic ovary syndrome |
| WHO: | = | world health organization. |
Acknowledgments
The authors would like to thank the embryology staff, nurses, and anesthesiologists for their help in collecting samples and recording data and the late Yefim Anbinder for laboratory assistance.
Declaration of interest: The authors report no declaration of interest.
Author contributions: Conceived and designed the experiment: AW, APL. Recruited patients and handled samples: AW, RBF, ML,YG. Laboratory analysis: DO. Wrote the manuscript: AW, APL. Reviewed and contributed to manuscript: DO, RBF, ML,YG.
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