Abstract
Objective
25(OH)D (Vitamin D) has been investigated for its role in the process of folliculogenesis and thus affects the quality of oocyte produced by in vitro fertilization. Our aims were to investigate the effects of 25(OH)D levels in follicular fluid, follicular estradiol level, successful fertilization rate in IVF treatment and the correlation between 25(OH)D levels in follicular fluid with the oocyte quality.
Design and Setting
This is an analytic observational study with cross-sectional design conducted between September–November 2018 in two fertility clinics at tertiary hospitals in Bandung, Indonesia.
Participants
The inclusion criteria were women aged 20–35 years who underwent controlled ovarian stimulation in IVF program with normal ovarian reserve and normal BMI. Eligible women were divided into two groups based on their 25(OH)D levels: low and high.
Results
There was a significant difference in oocyte quality (p = 0.03) and follicular estradiol levels (p = 0.02) between the two categories of 25(OH)D levels. High level of 25(OH)D has significantly higher level in comparison with the low level of 25(OH)D. No significant differences were found in terms of successful fertilization rate (p = 0.13). High level of 25(OH)D has higher successful fertilization rate compared to low level of 25(OH)D (71.8% vs 55.26%). A significant positive correlation between 25(OH)D level in follicular fluid and oocyte quality was also found (r = 0.32, p = 0.01).
Conclusion
Women with higher level of 25(OH)D are significantly more likely to have high-quality oocyte and follicular estradiol levels than those with low level of 25(OH)D, although there are no significant results for its relation to successful fertilization rate.
Keywords: Controlled ovarian stimulation, Estradiol level, Fertilization, Follicular fluid, In vitro fertilization, Oocyte quality, Vitamin D
Introduction
Oocyte quality plays an important role in IVF’s success. Several factors may influence its success rate, with female factors contributed up to 46.7% of unsuccessful pregnancy [1, 2]. Woman’s age, ovarian stimulation protocols, oocyte quality and maturity, number of embryos produced, preservation during the luteal phase, embryological laboratory environment, and the expertise of those involved contribute as the main factors for a successful pregnancy [3].
The follicular fluid has long been studied on its contents of various hormones and molecules that play a role in the autocrine and paracrine process during follicular development [4–6]. Aside from the FSH levels and its receptors in the follicle, there are several other substrates that have a major role in the process of folliculogenesis that affects the oocyte quality produced, one of which is vitamin D. Study has shown that vitamin D receptors can be found in ovary, suggesting that the hypoestrogenic conditions caused by the disrupted folliculogenesis process might be due to the absence of vitamin D in the follicles [7–9]. In recent years, abnormalities of vitamin D have been shown to cause detrimental effects on female’s reproductive organs, but number of researches conducted with human test subjects are limited [10, 11].
Researches have indicated that vitamin D can affect the process of folliculogenesis through several processes, such as increasing cell proliferation and suppressing the inflammatory process in the follicles [12, 13], suppressing the apoptosis process [12, 14], and increasing estrone, estradiol, and progesterone levels [13, 15, 16].
Therefore, these past studies have indicated that the binding of vitamin D in follicular fluid to its receptors on the granulosa cells in the ovarian follicle might play a role in various regulations of gene expressions that could affect the process of folliculogenesis and subsequently affect the quality of oocytes and the estradiol levels of produced follicular fluid [17–19]. Our present study aims to investigate the effects of different 25(OH)D levels in follicular fluid on oocyte quality, follicular estradiol level, and successful fertilization rate in IVF treatment as well as to determine the correlation between 25(OH)D levels in follicular fluid at the time of ovum pick-up with the oocyte quality obtained.
Materials and Methods
Design, Setting, and Study Population
This is an analytic observational study with cross-sectional design conducted between September–November 2018 in two fertility clinics at tertiary hospitals in Bandung, West, Java, Indonesia. We used follicular fluid collected from stimulated ovaries of patients during the ovum pick-up phase with a 36-h period given between the triggering of the ovaries and the pick-up phase. One follicle was considered as one observation unit.
Study Subjects
Women within the age range of 20–35 years old who underwent controlled ovarian stimulation in IVF program, normal ovarian reserve (AMH.1.2 ng/mL or AFC > 10), and normal BMI were included. Subjects with polycystic ovary syndrome, endometriosis, more than two oocytes found in one follicular fluid container, no oocyte found in a follicle, flushing solution contamination of ovarian fluid, and poor partner sperm parameters were excluded from this study as well as subjects who did not finish the treatment program.
All subjects were given thorough explanation on the study’s aims and procedures. Consents were obtained before commencing the study. The Health Research Ethics Committees from Faculty of Medicine, Universitas Padjadjaran and Dr. Hasan Sadikin Hospital Bandung approved the study protocol.
All patients underwent the controlled ovarian stimulation protocol using recombinant FSH, with the dose adjusted according to the patient’s characteristics. The ultrasonographic monitoring of follicles development were done from day 6 of menstrual cycle. After optimal follicle development were achieved, 250 µg of recombinant hCG were administered 34–36 h before scheduled oocyte pick-up procedure. During oocyte pick-up, each follicle was aspirated and follicular fluid was collected in separate containers, labeled, and tracked until the fertilization process. No follicular flushing was done. Blood-contaminated follicular fluid was not included in further assay.
Subsequently, the follicular fluid was centrifuged at 3000 rpm, separated into 2 aliquots of 0.5–1 mL each, and stored at a temperature of -2000C. The 25(OH)D levels in follicular fluid were measured using electro-chemiluminescence immunoassay (ECLIA) and CEA915Ge reagent kit (Cloud-Clone Corp) [20–23]. The follicular estradiol levels (pg/mL) were measured using enzyme-linked immunosorbent assay (ELISA) and E-EL-0065 reagent kit (Elabscience) [17, 24–27].
Oocytes extracted were denuded to assess its quality based on total oocyte scoring system’s six parameters: oocyte’s shape, oocyte’s size, the characteristics of ooplasm, the structure of perivitelline space, thickness of zona pellucida, and morphology of the polar body [28]. The fertilization was done using intracytoplasmic sperm injection (ICSI) and determined by appearance of two pronuclei 18–24 h after ICSI procedure.
Univariate analysis was done to describe the characteristics of the study subjects. To analyze the difference between follicular fluid estradiol level in two observation groups, the Mann–Whitney test was used since the data were not normally distributed. The differences between oocyte quality and fertilization rate were tested using chi-square test and the correlation between intrafollicular 25(OH)D levels and oocyte quality was tested using Spearman’s-rank test. The P value < 0.05 was considered statistically significant, with the statistical analysis being done using IBM SPSS version 21.
Results
Throughout the study period, 77 follicles were obtained. The study subjects’ characteristics are shown in Table 1. In this study, patients who were selected for their follicles as the materials in this study were adult female patients aged 20–35 years old who participated in the IVF program, met the requirements to be included in the study, had undergone a complete basic infertility examination, and had adequate ovarian reserves ((AMH level of > 1.2 ng/mL or AFC of > 5). Several studies have suggested that the role of 1,25 dihydroxyvitamin D in regulating the AMH levels and increasing the granulosa cell proliferation up to 6–8 mm in follicles that were incubated with 1,25-dihydroxyvitamin D [29].
Table 1.
Distribution of oocyte morphology assessment
| Parameters | Score | ||
|---|---|---|---|
| − 1 | 0 | 1 | |
| Morphology | 6 | 11 | 60 |
| Size | 8 | 11 | 58 |
| Cytoplasma | 7 | 9 | 61 |
| Zona pellucida | 15 | 8 | 54 |
| Perivitelline space | 22 | 12 | 43 |
| Polar body | 25 | 22 | 30 |
Overall 25(OH)D levels of follicular fluid obtained from this study were at deficiency levels with a media value of 13.7 ng/mL, slightly different from the study done by Pagliardini in Italy, where there were almost 70% of the infertility patients with vitamin D deficiency had a normal 25(OH)D levels [30]. Another study conducted in North Sumatra by Sari et al. found that 94.9% of women had vitamin D deficiency and only 5.1% were in the range of normal 25(OH)D levels with average of 17.71 ng/mL[31].
In this study, the overall levels of 25(OH)D of follicular fluid were at a deficiency level with a mean of 12.2 ng/mL and no follicles with normal 25 (OH)D levels were found. Hereafter, using the median value of 13.7 ng/mL, a sensitivity value of 56.25% and a specificity value of 76.92% were obtained. Therefore, the study group were divided into 2 groups: group A (low 25 (OH)D levels < 13.7 ng/mL) and group B (high 25(OH)D levels ≥ 13.7 ng/mL). Further analysis was carried out based on those classification groups (Table 2).
Table 2.
The distribution of oocyte quality in different follicular fluid 25(OH)D levels
| Oocyte quality | Observation group | p value | |
|---|---|---|---|
| A (25(OH)D < 13.7 ng/mL) |
B (25(OH)D > 13.7 ng/mL) |
||
| Good | 28 (74%) | 36 (92%) | 0.03 |
| Poor | 10 (26%) | 3 (8%) | |
x2: chi-square test
From the figure above, it can be observed that the polar body score for -1 has the highest number of oocytes with a prevalence rate of 32.5%, while 28.6% gets a score of 0 and only 38.9% gets the score of 1. From this study, 28.6% of oocytes were obtained from the incomplete oocyte maturation process and it was characterized by the poor polar body scores.
From Table 3, it can be concluded that the number of high-quality oocyte was more common in group B compared to group A (36 (92%) and 28 (74%), respectively) with statistically significant difference.
Table 3.
The comparison of the follicular fluid estradiol levels in both observation groups
| Follicular fluid estradiol levels | Observation group | Z-score | p value | |
|---|---|---|---|---|
| Group A (25(OH)D < 13.7 ng/mL) (n = 38) |
Group B (25(OH)D ≥ 13.7 ng/mL) (n = 39) |
|||
| Mean (SD) | 5106.7 (169.1) | 5164.8 (45.4) | − 2.4 | 0.02 |
| Median | 5153.3 | 5165.6 | ||
| Range (min–max) | 4146.6–5244.55 | 5087.40–5252 | ||
Mann–Whitney test
In Table 4, the difference of follicular fluid estradiol levels in both observation groups were shown. It can be concluded that the follicular fluid estradiol level was significantly higher in group B than in group A (5164.83 (45,41) pg/mL and 5106.65 (169.12) pg/mL, respectively).
Table 4.
The comparison of the successful fertilization rate in different follicular fluid 25(OH)D level
| Fertilization | Observation group | p value | |
|---|---|---|---|
| Group A (25(OH)D < 13.7 ng/mL) (n = 38) |
Group B (25(OH)D ≥ 13.7 ng/mL) (n = 39) |
||
| Failed | 17 (44.74%) | 11 (28.2%) | 0.13 |
| Succeeded | 21 (55.26%) | 28 (71.8%) | |
x2: chi-square test
As stated in the table above, the proportion of successful fertilization was higher in group B compared to group A (72% vs 55%), and the proportion of failed IVF treatment in group B was lower than group A (28% vs. 45%, respectively), although the differences showed statistically non-significant results.
A statistically significant correlation was also found (p = 0.01), although with a positive but weak correlation (r < 0.4). Therefore, it can be concluded that there was a statistically significant positive correlation between the 25(OH)D level of follicular fluid groups with total oocyte scores.
Discussion
In the past few years, several factors have been known to play relevant roles in achieving successful IVF treatment. It is expected that vitamin D could assist in the folliculogenesis process and increase the success of fertilization rate of IVF treatment [32, 33].
In this study, samples were collected from adult female patients aged 20–35 years old who participated in the IVF program and met the criteria requirements.
Overall follicular fluid 25(OH)D levels in this study were at deficiency levels, with a median value of 13.7 ng/mL. A study conducted in North Sumatra, Indonesia, by Sari et al. found that 94.9% of women had vitamin D deficiency and only 5.1% were in condition with normal 25(OH)D levels with an average value of 17.71 ng/mL [31, 32]. These findings raised questions about the standard distribution of vitamin D levels among Asian and Indonesian women in particular as well as to see whether the classification of vitamin D status that has been commonly known so far is consistent with the characteristics of Indonesian women.
The oocyte quality, embryo development and the success rate of IVF treatment are closely related. Until recently, the oocyte quality was deemed as one of the main predictor tools for pregnancy, thus its quality needs to be assessed. The oocyte quality criteria observed based on six parameters: oocyte’s shape, oocyte’s size, the characteristics of ooplasm, the structure of perivitelline space, thickness of zona pellucida, and morphology of the polar body and each parameter was divided into three groups (score − 1, score 0 or score + 1) [28, 33–35]. Therefore, poor oocyte quality might result in failed IVF treatment. In this study, there was a statistically significant positive correlation between the 25(OH)D level of follicular fluid groups with oocyte quality, even though the overall materials were in the category of vitamin D deficiency level.
Despite inconsistent results and controversy, our results are similar to the previous study in IVF patients with an average age range of 39.8 ± 5.6 years old where it stated that total oocyte scoring for the assessment of oocyte quality is one of the clinically useful tools to provide a prognostic picture of patients participating in the IVF program. Moreover, it’s also stated that administration of vitamin D supplementation for patients with deficiencies gave better results in the ovarian reserve quality markers measurement and the follicular dynamics [28].
In this study, researchers tried to assess the differences of oocyte quality in different 25(OH)D levels. However, after 25(OH)D examination of all follicular fluid samples, the results were all in the deficiency levels. Therefore, all study materials were separated into two groups based on the median value of 25(OH)D level in follicular fluid.
The proportion of good quality oocyte were higher in group B (25(OH)D level ≥ 13.7 ng/mL) compared with those in group A (25(OH)D level < 13.7 ng/mL). Furthermore, the proportion of low-quality oocyte was higher in the group A compared with group B. This supports the hypothesis that vitamin D plays an important role in the maturation process of oocytes. In folliculogenesis, vitamin D could trigger cell proliferation and suppress the inflammatory process in the follicles, suppressing the apoptosis process, and increasing the estron, estradiol, and progesterone levels. These findings are supported by Irani et al.’s research that found out the proportion of good quality oocyte was higher in groups with normal vitamin D levels compared to groups with low vitamin D levels [20, 21].
Estradiol is regarded to play a key role in women’s fertility processes. In this study, we found a significantly higher intrafollicular fluid estradiol concentration in group B compared with group A (5264.8 pg/mL vs 5106.6 pg/mL respectively, p = 0.02). This supports the assumption that intrafollicular vitamin D activity will promote the process of steroidogenesis. A research done by Parikh et al. discovered that an increase in progesterone production by 13%, an increase in estradiol by 9%, and an increase in estron by 21% in response to the vitamin D administration in adult female ovarian cell cultures which was the result of the steroidogenesis process. Therefore, this indicated that vitamin D is one of the essential factors for optimizing the process of steroidogenesis and gonadal function in women [23].
Vitamin D levels were suggested to have a detrimental effect on the development of endometrium and oocytes. However, in regards to the role of 25(OH)D levels from follicular fluid in fertilization, further in-depth researches are needed [29]. In this study, although there were no statistically significant differences between the two groups of 25(OH)D levels with successful fertilization rate, it could still be seen that the successful rate of fertilization in IVF treatment in group B was better than in group A.
The non-statistically significant outcome in this study (p = 0.13) shared some similar results with studies in Asian women which also demonstrated a contradictory correlation, where the successful rate of IVF was higher in women with low serum of vitamin D levels. A study by Estes et al. suggested that despite the low vitamin-D binding protein (VDBP) levels of follicular fluid in patient who failed the IVF treatment, it was still not certain that the serum vitamin D level or follicular fluid were closely related to the IVF treatment’s results [36].
The ICSI process on IVF program will bypass almost all of the natural fertilization processes starting from the penetration of cumulus cells to the fusion with oocytes. The fertilization process that cannot be replaced by ICSI is the activation of the oocytes and the subsequent cell division processes. In this study, the entire fertilization processes were done using the ICSI technique. Therefore, the non-statistically significant differences between the two-study groups were due to almost the entirety of vitamin D-influenced calcium metabolism was replaced by the ICSI process [37, 38].
The limitations for this study were the data of 25(OH)D levels in serum were not obtained, thus there was a mismatch in classifying vitamin D follicles using vitamin D serum parameters. Another limitation is the normal value of 25(OH)D in follicles were not known, so the cut-off to further classify the low and high groups of vitamin D levels were based on the median value of our subjects. Furthermore, all samples obtained were in vitamin D deficiency status. Therefore, further assessments to obtain the correlation between higher levels of 25(OH)D in follicular fluids with the oocyte quality are required for extensive researches.
Conclusion
Women with high level of 25(OH)D are significantly more likely than those with low level of 25(OH)D to have high-quality oocyte and follicular estradiol levels although there are no significant results for its relation to successful fertilization rate. Also, there is no positive correlation between 25(OH)D level in follicular fluid and oocyte quality obtained from the ovum pick-up.
Additional studies are needed to confirm the role and potential therapeutic benefits of vitamin D supplementation for a successful IVF treatment.
Acknowledgements
None.
Christofani Ekapatria
is an obstetrician and gynecologist who is specialized in fertility. He is one of the full time doctors in Siloam Hospitals Lippo Village and is currently pursuing doctoral degree in Padjajaran University Bandung.
Funding
None.
Availability of Data and Materials
The data that support the findings of this study are available on request from the corresponding author, CE. The data are not publicly available to respect participant’s privacy.
Declarations
Conflicts of interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Consent to Participate
All subjects were given thorough explanation of study aim and procedures, and consents were obtained before commencing the study.
Consent for Publication
All subjects have given consent to let the data obtained by the author to be published for scientific purpose.
Ethics Approval
The clinical trial ethics has been approved by the Health Research Ethics Committees from Faculty of Medicine, Universitas Padjadjaran and Dr. Hasan Sadikin Hospital Bandung.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author, CE. The data are not publicly available to respect participant’s privacy.