|Ahead of print publication
Serum homocystein and C-reactive protein in predicting outcome of in-vitro fertilization in infertile women with polycystic ovary syndrome
Kingsley C Onwumere1, Bolanle O P Musa1, Abimbola O Kolawole2, Nkeiruka Ameh3, Ibrahim Wada3, Christopher Danga3, Abdurrahman Ahmad EL-Fulaty1, Bamgbopa Kehinde4
1 Department of Medicine, Ahmadu Bello University, Zaria, Nigeria
2 Department of Obstetrics and Gynaecology, Ahmadu Bello University, Zaria, Nigeria
3 Department of In Vitro Fertilisation Department, Nisa Premier Hospital, Abuja, Nigeria
4 Department of Obstetrics and Gynaecolgy, NNPC Medical Services Limited, Abuja, Nigeria
|Date of Submission||04-Jan-2022|
|Date of Acceptance||15-Feb-2022|
|Date of Web Publication||27-Jun-2022|
Kingsley C Onwumere,
Department of Medicine, Ahmadu Bello University, Zaria
Source of Support: None, Conflict of Interest: None
Background: Polycystic ovarian syndrome (PCOS) is the most common cause of anovulatory infertility. Hyperhomocysteinemia (HHcy), is common in PCOS and reportedly causes poor oocyte quality and recurrent pregnancy loss. The aim of this report is to assess serum concentrations of Hcy and its association with C-reactive protein (CRP) levels in patients with PCOS undergoing in-vitro fertilization (IVF) treatment. Materials and Methods: Eighty-five women of reproductive age 25–35 years were included in this study, comprising 30 infertile women with PCOS (group 1), 30 infertile women without PCOS (group 2), and 25 matched apparently healthy women without infertility or PCOS (group 3). The serum Hcy and CRP levels were measured using the TOSOH AIA 360 enzyme immunoassay method and Raytor6000 Semi-Autoanalyzer, respectively. Results: The median CRP values were 15.13 (4.43–21.77 mg/L), 3.42 (2.50–8.04 mg/L), and 1.45 (0.97–3.51 mg/L) for Group 1, Group 2, and Group 3, respectively. The median Hcy values were 19.75 (15.50–39.98 μmol/L), 10.00 (9.38–10.92 μmol/L), and 10.00 (8.93–10.93 μmol/L) for Group 1, Group 2, and Group 3, respectively. The differences between Group 1 and Group 2, and between Group 1 and Group 3 median Hcy values were significant for both CPR and Hcy (P < 0.0001). There was a significant association between embryo qualities and the groups. Similarly, there were more of higher (Grades 4 and 5) embryo qualities in Group 3. Conclusion: Serum Hcy and CRP levels estimation should be included in the evaluation and management of patients with PCOS undergoing in-vitro fertilization.
Keywords: C-reactive protein, homocystein, IVF, PCOS
|How to cite this URL:|
Onwumere KC, Musa BO, Kolawole AO, Ameh N, Wada I, Danga C, EL-Fulaty AA, Kehinde B. Serum homocystein and C-reactive protein in predicting outcome of in-vitro fertilization in infertile women with polycystic ovary syndrome. Afr J Infertil Assist Concept [Epub ahead of print] [cited 2022 Sep 26]. Available from: https://www.afrijiac.org/preprintarticle.asp?id=348368
| Introduction|| |
Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features, which serve as the central diagnostic criteria. The polycystic ovarian syndrome is the most common cause of anovulatory infertility.
Homocysteine (Hcy) is an endogenous amino acid, containing a free thiol group, which in healthy cells is involved in methionine and cysteine synthesis/re-synthesis., Oxidative stress caused by the relentless formation of free radicals within an environment without antioxidant balance is generated during oxidation of the free thiol group of Hcy. Oxidation of Hcy may induce the subsequent oxidation of proteins, lipids, and nucleic acids. This oxidative stress (OS) affects a variety of physiological processes such as folliculogenesis, oocyte maturation, endometrial cycle, luteolysis, implantation, and embryogenesis in the female reproductive tract, thus influencing reproductive outcome. Although Hcy is not harmful at normal levels, at abnormally high concentrations (especially during pregnancy), it can cause a condition called hypercoagulability, which can cause a higher risk of heart attack and stroke, and possibly endanger the baby during pregnancy.
C-reactive protein (CRP), an acute phase protein belonging to the pentraxin family of proteins, increases 1000-folds or more in concentration in blood during the occurrence of an injury, inflammation, or tissue death. PCOS state is a low-grade chronic inflammatory state and may stimulate the immune response, increasing inflammatory factors such as CRP.
Hyperhomocysteinemia (HHCY), a common finding among patients with PCOS, has been reported to have some detrimental effects including low-grade inflammatory response, poor oocyte quality, and recurrent pregnancy loss.,, Deranged Hcy levels could be a hidden infertility factor resulting in in-vitro fertilization (IVF) treatment failure that may be encountered in patients with PCOS.
There are only a few studies on the relationship between HHCY and anovulatory infertility., The aim of this study is to assess the relationship between IVF treatment outcomes and CRP with serum levels of Hcy in patients with PCOS. The hypothesis is that there is no difference in serum Hcy and CRP levels in patients with PCOS when compared with control groups. It is hoped that the findings will provide information that facilitates the development of “immunological agents” targeting Hcy in infertile women with PCOS.
| Materials and Methods|| |
This is a cross-sectional study involving women aged 25–35 years attending infertility clinic at Nisa Premier Hospital, Abuja, Nigeria, from July 2017 to August 2018. The women were in three groups as follows:
Group 1: infertile women with PCOS scheduled for IVF;
Group 2: infertile women without PCOS scheduled for IVF;
Group 3: apparently healthy women without infertility or PCOS scheduled for IVF for genetic reasons.
Inclusion criteria for group 1 were oligo-ovulation or anovulation manifested by oligomenorrhea or amenorrhea, hyperandrogenism, and absence of apparent infections.
Inclusion criteria for groups 2 and 3 were absence of PCOS and apparent absence of infection. Exclusion criteria were women already on drugs such as methotrexate and metformin, apparent presence of infection, nutritional deficiencies (vitamins B6, B12, folate), and sickle cell anemia, and systemic lupus erythematosus (SLE).
A calculated sample size of 25 was obtained for each group (increased to 28 to allow for attrition of 10%). Ultimately, 85 women were recruited into the study consisting of 30 in group 1, 30 in group 2, and 25 in group 3.
Demographic information was obtained from all study participants and their full blood count ascertained.
Blood sample collection and analysis
Blood samples were taken on day 2 or day 3 of the menstrual cycle before the IVF procedure started. After explaining the procedure to the patient, the site of venepuncture was identified. A tourniquet applied and the site sterilized with methylated spirit. Five mL of blood was taken in the syringe; 3 mL of the blood was transferred into a plain bottle, whereas 2 mL was transferred into an ethylenediaminetetraacetic acid (EDTA) bottle and gently mixed. The EDTA sample was stored in a fridge at a temperature of 2–8oC until analysis for full blood count. The blood in the plain bottle was allowed to clot for 60 min. Then the sample was spun for 3 min at 3000g to separate the serum from the cells. The separated serum was stored frozen at a temperature of −20oC until analysis for Hcy and CRP. The serum Hcy and CRP levels were measured using the TOSOH AIA 360 Enzyme Immunoassay method and Rayto6000 Semi-Autoanalyzer, respectively.
Each group was started on daily subcutaneous injections of 150 IU recombinant follicle-stimulating hormone (FSH), on the second day of a spontaneous cycle (Puregonen was started). Daily subcutaneous administration of 0.25 mg gonadotropin-releasing hormone antagonists (Orgalutran®, NV Organon) was started when at least one follicle measures ≥14 mm. To induce final oocyte maturation, a single dose of 10,000 IU human chorionic gonadotrophin sc (hCG, Pregnyl®, NV Organon) was administered as soon as the largest follicle reached at least 18 mm in diameter and at least one additional follicle of >15 mm is present. Oocyte retrieval was carried out 35 h after hCG injection by transvaginal ultrasound-guided puncture of the follicles. Immediately prior to this procedure, the diameter of the leading follicle in both the right and left ovaries was measured in three dimensions.
Grading of egg quality
Egg and embryo quality were categorized on a scale 1–5, in which grade 1 is poor, grade 2 is fair, grade 3 is good, grade 4 is very good, and grade 5 is excellent.
Evaluation of pregnancy rate
Pregnancy was assessed by carrying out serum pregnancy testing and quantitative beta-hCG for monitoring the pregnancy.
Ethical approval was obtained from Nisa Hospital Ethical and Clinical Research Committee. Informed consent was obtained from each study participant after adequate counseling.
Data analysis was performed using Analyse-it®, version 4.6, Method Evaluation Statistical Software for Microsoft Excel. Numerical averages such as age, serum Hcy level, and CRP levels are presented as mean and standard deviation or median and ranges. Qualitative variables were analyzed using the χ2 test to determine statistical association, whereas Student’s t-test was used to compare quantitative variables. Level of statistical significance was set at 95% confidence interval and P ≤ 0.05.
| Results|| |
The demographics of the three groups are summarized in [Table 1]. The groups were matched for age, FSH, l uteinizing hormone (LH), prolactin levels, and hematological parameters.
CRP levels and Hcy levels
The median CRP values (mg/L) were 15.13 (4.43–21.77), 3.42 (2.50–8.04), and 1.45 (0.97–3.51) for groups 1–3, respectively. Further analysis revealed significant differences between group 1 and group 2 (P = 0.0001) and also between group 1 and group 3 (P < 0.0001) [Table 2].
|Table 2: Comparison of CRP and Hcy levels among three groups of women undergoing IVF|
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The median Hcy values (µmol/L) were 19.75 (15.50–39.98), 10.00 (9.38–10.92), and 10.00 (8.93–10.93) for groups 1–3, respectively. Further analysis revealed significant difference between Group 1 and Group 2 (P < 0.0001) and between Group 1 and Group 3 (P < 0.0001) [Table 2].
There was a weak correlation between CRP and Hcy levels in groups 1 (r = 0.34; 34%, P = 0.07) and 2 (r =0.25; 25%, P = 0.17). In group 3, there was a moderately high level of anti-correlation (r =−0.64) between CRP and Hcy, which was significant (P<0.01) [Figure 1][Figure 2][Figure 3].
There were higher (4 and 5) embryo qualities in group 3 compared with groups 1 and 2, which had lower embryo qualities (1, 2, and 3). The result of the χ2 test (Appendix) revealed a significant association between embryo qualities and the groups.
Relationship between Hcy levels and oocyte quality
There were higher (4 and 5) egg qualities in group 3. None of the groups had the lowest egg quality of grade 1. There was a significant association between egg qualities and the groups (P < 0.0001) [Figure 4] and [Figure 5].
The highest concentration of Hcy (60.40 ± 40.66) was in the lowest egg quality (grade 2) in group 1. The lowest concentration of Hcy (8.74 ± 2.60) was recorded in the highest egg quality (grade 5) in group 3. Multiple comparisons by the Kruskal–Wallis test showed significant difference (P < 0.0033) in Hcy concentrations in women with low quality eggs; for grade 2 in Group 1, all other comparisons were not significant (P > 0.05) [Table 3].
|Table 3: Egg quality and Hcy concentration in three groups of women undergoing IVF|
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The pregnancy rates were 7 (23%) in group 1, 8 (27%) in group 2, and 16 (64%) in group 3. The relationship between PCOS and success of IVF was not statistically significant (P = 1.000). The relationships between groups 1 and 3 (P = 0.0056) and between groups 2 and 3 (P = 0.0122) in the success of IVF were significant [Table 4].
|Table 4: Relationships between IVF outcome in three groups of women undergoing IVF|
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Pregnancy outcome and Hcy levels
Comparing the Hcy concentrations in the groups between those with successful IVF and those with unsuccessful IVF shows a progressive relationship but there was no significant difference between the Hcy values in any group [Table 5].
|Table 5: Hcy concentration and pregnancy outcome in three groups of women undergoing IVF|
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| Discussion|| |
PCOS is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features. Reproductive health issues related to PCOS include abnormal uterine bleeding, endometrial abnormalities, infertility, acne, hair growth, and weight gain. HHCY, a common finding among PCOS, has been reported to have some detrimental effects on female reproduction. PCOS state, as a low-grade chronic inflammatory state, may stimulate the immune response, increasing inflammatory factors such as CRP. HHCY, a common finding among patients with PCOS, has been reported to have some detrimental effects on female reproduction such as low-grade inflammatory response, poor oocyte quality, and recurrent pregnancy loss.,, Deranged Hcy levels could be a hidden infertility factor resulting in IVF treatment failure commonly encountered in patients with PCOS. However, such relationship is poorly understood.
In the present report, serum levels of Hcy were found to be higher among infertile patients with PCOS compared with the matched control groups, as previously reported.,, Such HHCY among PCOS has been reported to induce low-grade inflammatory response, poor oocyte quality, and recurrent pregnancy loss.,, In our study, the serum levels of CRP were also found to be higher in infertile patients with PCOS. Other reports have indicated the presence of low-grade chronic inflammation in patients with PCOS.,,
Elevated circulating homocysteine or HHCY is known to be associated with biotoxicity and has been implicated as an emerging risk factor for several diseases such as unexplained female infertility. Immunologically, high homocysteine has been shown to induce vascular inflammation by enhancing the expression of proinflammatory cytokines, such as monocyte chemoattractant protein 1, which regulates migration and activation of monocytes/macrophages, and interleukin-8 (IL-8), which is an important chemoattractant for neutrophils and T-lymphocytes., High levels of homocysteine decrease the bioavailability of NO, one of the major endothelium-dependent vasodilators that is produced by the endothelial isoform of eNOS. This effect is caused either by an accelerated oxidative inactivation of NO and/or eNOS or by an increase in serum asymmetric dimethylarginine, an endogenous inhibitor of eNOS.
A correlation analysis between Hcy and CRP in the serum samples of the groups studied showed a weak correlation in infertile women with PCOS and without PCOS, but there was a moderately high level of anti-correlation in fertile women, which was significant. Patients with elevated levels of CRP have been shown to elicit impaired endothelium-dependent vasodilatation, suggesting that CRP may be a useful clinical tool for endothelial vasomotion. CRP may also directly promote monocyte activation by stimulating the release of cytokines, such as IL-1b, IL-6, and tumor necrosis factor-α. In addition, one report has indicated a possible association between inflammatory response and HHCY with incidence of the latter being an increasingly frequent finding in women with PCOS. Hypofibronolysis associated with high levels of another acute phase protein, plasminogen activator inhibitor-1 (PAI-1) in women with PCOS, and recurrent pregnancy loss (RPL) has been reported and indicates that the effects of elevated PAI-1 may be aggravated by elevated Hcy, eventually causing thrombosis.
In the present report, it has been demonstrated that high serum concentrations of Hcy negatively affect egg quality with the highest concentration of Hcy associated with the lowest egg quality (grade 2) in infertile women with PCOS. The lowest concentration of Hcy was recorded in the highest egg quality (grade 5) in fertile women without PCOS. One report indicated that some women have a tendency to produce higher-than-ideal levels of Hcy in their bodies which can affect fertility and IVF success. High Hcy levels in follicular fluid have also been reported to cause decreased cell division and high fragmentation in embryo cultures, resulting in a decrease in oocyte and embryo quality. Oocytes exposed to low Hcy concentration present better quality and higher degree of maturity and show a correlation between follicular fluid Hcy concentration and oocyte maturity. The plasma Hcy levels correlate with follicular fluid Hcy levels. High plasma Hcy levels may lead to oxidative stress, and this may in turn affect the ovulation process and eventually cause infertility. Szymański and Kazdepka-Ziemińska reported that mature oocyte count is high in patients with low Hcy levels and there was a relationship between Hcy levels and oocyte maturity. An inverse association between follicular fluid Hcy levels and oocyte and embryo quality has been reported. It has been reported that more oxygen radicals are found in follicular fluid of women who conceive with IVF compared with women who did not conceive with IVF, and the detrimental effects of high Hcy levels are due to free radicals induced by Hcy oxidation.
The distribution of IVF outcome in the three groups indicates that fertile women without PCOS had the lowest average concentration of Hcy and the highest pregnancy ratio. Although this relationship was progressive, it did not reach statistical significance. This is similar to another report indicating that implantation and pregnancy rates of women with low follicular Hcy levels were higher when compared with women with high follicular fluid Hcy levels. One report indicates that successful pregnancy outcome is highly dependent on satisfactory placental development and sustained placental function that may be compromised by increased thrombosis caused by microthrombi in the vessel bed of the placenta. These microthrombi may cause multiple placental infarctions and subsequently maternal complications of pregnancy. The report suggests that RPL is associated with an increased risk of thrombosis caused by heritable thrombophilic defects, or HHCY, or combinations of these two pathologic conditions.
| Conclusion and Recommendation|| |
This study demonstrated a significant relationship between Hcy and CRP levels and PCOS in women who are undergoing IVF. The Hcy concentration was highest in infertile women with PCOS and lowest egg quality, and the lowest concentration was in fertile women without PCOS and highest egg quality. The IVF pregnancy rate was highest in fertile women without PCOS. Overall, infertile women with PCOS had higher concentration of Hcy and CRP and lowest egg quality and IVF pregnancy rate, indicating a role for inflammation in poor pregnancy outcome and infertility associated with PCOS. It is recommended that serum Hcy and CRP evaluation should be included in the evaluation and management of patients with PCOS undergoing in IVF to facilitate planning and improvements in IVF outcomes.
This study demonstrated the occurrence of PCOS is associated with elevated serum CRP levels indicating the presence of low-grade inflammatory response in patients undergoing IVF treatments. A high Hcy level is associated with poor egg and Embryo quality in PCOS patients undergoing IVF. Hcy levels in non-PCOS and fertile women undergoing IVF procedures are generally within the normal range (6.0–15.0 µmol/L) and were found to be associated with positive IVF outcomes.
It is recommended that serum Hcy and CRP evaluation should be included in the evaluation and management of patients with PCOS undergoing IVF to facilitate planning and improvements in IVF outcomes.
Further studies are required with larger sample sizes to validate the findings of this study. A limitation of this report is the inability to exclude confounders such as infections, nutritional deficiencies (e.g., B6, B12, folate), MTHFR gene mutations (especially the C677T variation), sickle cell anemia, and SLE in the three groups of patients.
Special appreciation to God Almighty and to all the co-authors.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]