Oocyte Maturation and Fertilization-Related Outcomes Following a Pro-Fertility Diet Combined with Omega-3 Supplementation in Women Undergoing Assisted Reproductive Technology: A Secondary Analysis of a Randomized Controlled Trial

Abstract

Purpose

Oocyte developmental competence is a key determinant of IVF success, but randomized evidence on multi-component dietary interventions is limited. This paper reports prespecified secondary outcomes of a registered randomized trial (IRCT20130903014551N13) that examined whether a Pro-Fertility Diet (PFD) combined with omega-3 supplementation affects oocyte-related outcomes in women undergoing IVF/ICSI.

Methods

In this randomized, parallel-group, single-blind (outcome assessors) trial at Hazrat Maryam Fertility Center (Isfahan, Iran), 180 infertile women aged 18–42 years were allocated (stratified block randomization by age and BMI) to a PFD plus omega-3 supplementation or to standard dietary advice for 8 weeks before IVF/ICSI. Embryo-related outcomes and fertilization rate from this trial have been published previously. The outcomes reported here for the first time are antral follicle count (AFC), cumulus–oocyte complexes (COCs), metaphase II (MII) oocytes and two-pronuclear (2PN) zygotes, analyzed in 167 women with available outcome data (complete-case analysis; 84 intervention, 83 control). Count outcomes were analyzed using Poisson regression with robust standard errors and fertilization rate (2PN per MII oocyte) using binomial regression, both adjusted for age and BMI.

Results

Of the 180 randomized participants, 167 women (84 in the intervention group and 83 in the control group) were included in the complete-case analysis of oocyte counts. Compared with controls, the intervention group had higher MII oocyte counts (adjusted IRR = 1.16, 95% CI: 1.01–1.33; P = 0.035) and 2PN zygote counts (IRR = 1.38, 95% CI: 1.17–1.61; P < 0.001), as well as higher odds of fertilization (OR = 1.92, 95% CI: 1.41–2.63; P < 0.001). No statistically significant differences were observed for AFC or COC counts. After adjustment for multiple comparisons using the Holm and Benjamini–Hochberg procedures, the difference in 2PN zygote counts remained statistically significant (adjusted P < 0.001 with both methods), whereas the difference in MII oocyte counts did not (Holm-adjusted P = 0.104; BH-adjusted P = 0.069).

Conclusions

In this exploratory secondary analysis, the combined nutritional intervention was associated with a higher 2PN zygote count, which remained statistically significant after correction for multiple comparisons. Although a higher MII oocyte count was observed in the initial adjusted analysis, this finding did not remain statistically significant after multiplicity adjustment. Further adequately powered randomized controlled trials are needed to confirm these findings.

Introduction

Infertility is defined as the inability to achieve pregnancy after 12 months of regular, unprotected sexual intercourse and is associated with significant psychological, social, and financial consequences for affected couples.¹ The global prevalence of infertility has risen steadily, increasing from 42 million cases in 1990 to 48.5 million in 2000, with projections estimating that 60 million couples will be affected by 2030.² Assisted Reproductive Technology (ART) serves as the primary treatment for infertility, with in vitro fertilization (IVF) being the most widely used ART approach, involving both medical and pharmacological interventions.³ Despite the widespread application of IVF—with approximately 2.6 million cycles performed annually worldwide and over 500,000 infants born via this method⁴—the live birth rate per cycle remains modest, ranging between 30% and 40%.³ Given the moderate success rates, high treatment costs, and limited accessibility in certain regions, optimizing modifiable factors influencing ART outcomes is crucial. Among these factors, oocyte quality plays a pivotal role in IVF success,⁵ with key determinants including advanced maternal age, elevated body mass index (BMI), and environmental factors such as dietary intakes.⁶

The evidence for the Pro-Fertility Diet (PFD) and for omega-3 fatty acids differs in nature and strength and is therefore considered separately. The PFD was derived by Gaskins et al from a prospective cohort of women undergoing ART and combines a dietary pattern (higher intake of whole grains, soy, low-fat dairy, seafood and low-pesticide fruit and vegetables) with supplemental folic acid, vitamin B12 and vitamin D.⁷ Observational studies link adherence to this pattern with more favorable ART outcomes and ovarian reserve markers,^7,8 but its effect on oocyte-related outcomes has not been tested in randomized trials. Separately, omega-3 fatty acid intake has been associated with fecund ability,⁹ and small randomized trials of omega-3 sources (fish oil, flaxseed oil) before IVF/ICSI have reported more MII oocytes or higher fertilization rates,^10,11 although a systematic review judged the evidence limited and heterogeneous.¹²

Although adherence to the PFD has been associated with favorable reproductive outcomes in observational studies, evidence from randomized controlled trials evaluating its effects on oocyte-related outcomes remains limited. Evidence regarding individual nutrients and supplements, including omega-3 fatty acids, has also suggested potential roles in reproductive function; however, these findings are based on evidence concerning individual dietary components rather than the PFD as a whole. Because the PFD incorporates multiple dietary components that may be relevant to reproductive health, combining the dietary pattern with selected nutritional supplements may represent a pragmatic approach to pre-IVF nutritional preparation. However, the independent or synergistic effects of these components have not been established. Therefore, the present randomized controlled trial was designed to evaluate the effects of a PFD combined with omega-3 fatty acid supplementation and other nutritional supplements on oocyte maturation in women undergoing IVF.

Methods

Study Design and Participants

This parallel, randomized, single-blind (outcome assessors) clinical trial was designed to examine the effect of a PFD intervention on oocyte maturation in women with infertility undergoing IVF treatment. The present manuscript reports previously unpublished oocyte-related outcomes from the parent randomized controlled trial, including antral follicle count (AFC), cumulus–oocyte complexes (COCs), metaphase II (MII) oocytes, and two-pronuclear (2PN) zygotes. Embryo-related outcomes and fertilization rate have been reported in a previous publication from the same trial,¹³ Fertilization rate is also presented in the current manuscript to provide context for interpreting the oocyte-related findings and is not considered a novel outcome of this analysis. The current analysis focuses on distinct outcome measures that were not included in the previous publication. This study was performed from June 2024 to June 2025 at the Hazrat Maryam Fertility Center of Isfahan University of Medical Sciences, Isfahan, Iran. Eligibility criteria for participation was determined by a gynecologist specialized in infertility treatment who identified candidates for ART, specifically IVF or intracytoplasmic sperm injection (ICSI). Women were included in this study if there were 18–42 years old with BMI=18.5–30 kg/m². These exclusion criteria were defined for this study: current or previous use (within the past 3 months) of oral contraceptives, insulin-sensitizing or anti-diabetic medications, weight-loss drugs, vitamin/mineral supplements, or other dietary supplements; diagnosis of chronic diseases or endocrine disorders including diabetes and glucose intolerance; history of more than two unsuccessful ART cycles; adherence to specific dietary regimens or structured exercise programs; tobacco or alcohol use; presence of uterine or tubal disorders causing infertility; consumption of fatty fish more than Once a week; any change in dietary pattern or physical activity for any reason; Initiation of a new medication or a change in the dosage of current medications that could affect the study outcomes.

The study was approved by the Ethics Committee of Isfahan University of Medical Sciences (ethics approval code: IR.ARI.MUI.REC.1402.091) and was registered in the Iranian Registry of Clinical Trials (URL: https://irct.ir/trial/71704; registration ID: IRCT20130903014551N13). Written informed consent was secured from all participants following a thorough explanation of the study’s aims and assurance of data confidentiality.

Changes to Trial Protocol

No changes were made to the trial protocol after commencement.

Sample Size

The sample size was calculated based on the pregnancy rate using a standard formula for comparing proportions. A conservative approach was applied, considering a control group success rate of 12.4% and an intervention group success rate (π₂) of 35.2%, with a two-sided type I error (α) of 0.05 and a power (1−β) of 0.90. Assuming equal group sizes (allocation ratio=1), the required sample size was estimated at 71 participants per group.¹⁴ To accommodate an anticipated 20% dropout rate, the sample size was increased to 90 participants (180 participants) per group. The present study reports oocyte-related secondary outcomes that were prespecified as part of the original randomized controlled trial. The sample size of the parent trial was calculated a priori based on pregnancy rate as the primary outcome. All oocyte-related outcomes examined in the present analysis were included in the original study protocol and were assessed within the same randomized study population. Therefore, no separate sample size calculation was performed specifically for these secondary outcomes.

Randomization and Blinding

Stratified block randomization was performed according to age (≤35 years and >35 years) and BMI category (BMI <25 kg/m² and 25≤BMI<30 kg/m²). Within each stratum, permuted block randomization with a fixed block size of 4 was used. The randomization sequence was generated using the Sealed Envelope online tool (https://www.sealedenvelope.com/simple-randomiser/v1/lists). The statistical consultant held the randomization list and communicated participants’ group assignments.

Blinding of participants was not feasible because the intervention involved specific dietary education and supplementation without matching placebos. Participants were not informed of the study hypothesis or which regimen was considered experimental. The gynecologist who assessed AFC and the embryologists who assessed COCs, MII oocytes, and 2PN zygotes were blinded to treatment allocation. The statistician was not blinded to group allocation.

Intervention and Comparator

Participants were randomly assigned to either the intervention group, which received a structured PFD combined with daily omega-3 supplementation, or the control group. The dietary intervention was implemented for eight weeks prior to the ART cycle—a window selected to allow sufficient time for potential diet-related effects on gamete maturation, while avoiding delays in treatment that could negatively influence adherence.¹⁵ Evidence from animal research indicates that dietary modifications during the peri-implantation period may shape early embryonic development.¹⁵ To ensure comparable energy intake, individual caloric requirements were estimated using the Harris–Benedict equation, and macronutrient distribution was standardized to 15–20% protein, 25–30% fat, and 55–60% carbohydrates.⁷ A registered dietitian prepared individualized weekly 7-day meal plans for all participants. The design of the PFD and the nutrient supplementation protocol was based on the framework proposed by Gaskin et al;⁷ moreover, findings from Alousi et al demonstrated that a daily dose of 1 g omega-3 can improve the follicle-to-retrieved oocyte ratio, increase the number of metaphase II oocytes, enhance fertilization rates, and promote higher-quality (grade I) embryos.¹⁰ The PFD was structured to optimize the intake of nutrients associated with reproductive outcomes.⁷ It emphasized at least four daily servings of low-pesticide fruits and vegetables (eg, prunes, onions, corn, cauliflower, cabbage, oranges, apple juice, grapefruit, tomato paste, melon, bananas, eggplant, zucchini, broccoli, carrots, lettuce), while limiting high-pesticide produce (such as tomatoes, squash, apples, peaches, green beans, grapes, raisins, potatoes, pears, spinach, plums, strawberries, celery, and bell peppers) to no more than one serving per day.⁷ Refined grains were replaced with whole grains; participants were advised to consume at least 2.5 servings of low-fat dairy daily; one serving of soy products was included each day (≈30 g soy, equivalent to 430 g soy milk or 50 g isolated soy protein); and the use of disposable plastic food containers was minimized.⁷ The diet also provided daily intakes of 1 mg folic acid, 500 µg vitamin B12, and 1000 IU vitamin D to ensure adequate levels of nutrients vital for oocyte competence and early embryogenesis.⁷ In addition to the PFD, participants received a daily 1000 mg omega-3 supplement derived from cold-water fish oil, containing approximately 180 mg EPA and 120 mg DHA (Nexus Pharmaceutical). Vitamin B12 and vitamin D supplements were manufactured by Nik Akhtar Arya under Health Aid UK authorization, and folic acid tablets were produced by Iran Darou. Women in the control group were provided with general nutrition recommendations based on the Healthy Eating Plate model,¹⁶ accompanied by written and verbal guidance to reduce consumption of processed meats and saturated or trans fats. They also received 500 µg of folic acid daily. In both the intervention (1 mg/day) and control (500 µg/day) groups, the folic acid supplement consisted of pteroylmonoglutamic acid, the conventional synthetic form of folic acid, rather than 5-methyltetrahydrofolate. At baseline, all participants completed demographic, lifestyle, and medical history questionnaires. A three-day dietary recall and anthropometric assessments were conducted both before and after the 8-week intervention. Fasting blood samples were taken at the same time points in both groups to measure serum 25(OH)D, folate, and vitamin B12 levels as objective markers of adherence. Compliance with supplementation was monitored through the return of empty packages and weekly follow-ups via phone or an online platform, while adherence to the dietary plan was assessed weekly when new meal plans were delivered. Throughout the study, participants continued their routine gynecologic medications as prescribed, and any changes were documented.

Harms

No adverse events or harms were reported by participants during the study period.

Assessment of Variables

Dietary Intake and Physical Activity Levels

Physical activity levels were assessed at both baseline and the end of the study using the International Physical Activity Questionnaire (IPAQ), with data converted to metabolic equivalent (MET)-minutes per week according to standard guidelines. Dietary intake was assessed using 3-day food records collected at two time points—once at baseline and once at the end of the intervention—resulting in a total of 6 days of dietary data per participant. Dietary data were analyzed using Nutritionist IV (N4) software.

Anthropometric Measurements

Anthropometric assessments were conducted at the first visit and at the end of the intervention. Weight was measured using a Seca mechanical scale with an accuracy of 0.1 kg, while participants wore minimal clothing and no shoes. Height was measured using a Seca stadiometer with an accuracy of 0.1 cm. BMI was calculated by dividing weight in kilograms by height in meters squared (kg/m²).

Assessment of Oocyte-Related Outcomes

Clinical outcomes including AFC, number of COCs retrieved, number of MII, Number of 2PN zygotes, and fertilization rate were measured at the end of the intervention and during the ART cycle. AFC was assessed by an experienced gynecologist using transvaginal ultrasound on the third day of the follicular phase. COC, MII oocytes, and 2PN were assessed by an embryologist. The fertilization rate was determined 17 to 20 hours after insemination, based on the ratio of oocytes with 2PN to the total number of MII oocytes.¹⁷ Intracytoplasmic sperm injection (ICSI) was performed for all participants in both study groups in accordance with the fertility center’s routine clinical protocol. Thus, the fertilization method was consistent across the intervention and control groups.

Biochemical Assessment

Because vitamin B12, folate, and 25-(OH)D exhibit minimal short-term post-prandial variation, non-fasting sampling does not materially affect measurement accuracy. Serum concentrations of vitamin B12, folate, and 25-hydroxyvitamin D were assessed using sensitive and standardized analytical methods to ensure the accuracy and reliability of the results. Vitamin B12 and folate were quantified via electrochemiluminescence immunoassay (ECLIA), a method recognized for its high precision, with intra-assay coefficients of variation below 5% and detection limits of 30 pg/mL and 0.5 ng/mL, respectively. These parameters allowed for reliable detection across a wide range of nutritional statuses.¹⁸ For vitamin D, total 25(OH)D was measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS), which is considered the reference method due to its high specificity. The procedure included derivatization and calibration against NIST SRM 972a, with an intra-assay CV below 4% and a detection limit of 2 ng/mL. This ensured accurate assessment even at low serum concentrations.¹⁹

Statistical Analyses

Statistical analysis was performed using SPSS version 20 and R/Python (statsmodels) for generalized linear models. Continuous variables are presented as mean ± standard deviation (SD) or median (Q1–Q3), and categorical variables as frequencies (percentages). Baseline characteristics were compared between groups using the independent-samples t-test for continuous variables and the chi-square test for categorical variables. Within-group changes in dietary intake and serum micronutrients were assessed using paired t-tests, and between-group differences in these variables were assessed using ANCOVA adjusted for baseline values. Oocyte-related outcomes were measured once during the ART cycle and therefore had no corresponding baseline measurements. AFC, COC, MII oocyte, and 2PN zygote counts were analyzed using Poisson regression with a log link and robust (sandwich) standard errors, adjusting for age and BMI. Adjusted effects are reported as incidence rate ratios (IRRs) with corresponding 95% confidence intervals. Fertilization rate was analyzed using binomial logistic regression, modeling the number of 2PN zygotes relative to the number of MII oocytes for each participant, with robust standard errors and adjustment for age and BMI. Results are reported as adjusted odds ratios (ORs) with corresponding 95% confidence intervals. This analysis included 166 participants because one record with a 2PN count exceeding the MII oocyte count was excluded. All statistical tests were two-sided, with P < 0.05 considered statistically significant in the nominal analyses. Given the exploratory nature of the secondary outcomes, no formal adjustment for multiple comparisons was applied in the primary analyses. To assess the robustness of the findings to multiple testing, sensitivity analyses were performed using the Holm step-down procedure and the Benjamini–Hochberg (BH) false discovery rate procedure across the four newly reported oocyte-related outcomes (AFC, COC, MII, and 2PN). The multiplicity-adjusted P values are presented in Supplementary Table S1.

Analyses were conducted using a complete-case approach (n = 167), and missing outcome data were not imputed. All oocyte-related secondary analyses were interpreted as exploratory.

Results

At the beginning of the study, 240 women with infertility were assessed for eligibility. Of these, 27 did not meet the inclusion criteria, and 33 declined to participate. Consequently, 180 women were randomized into two equal groups (n = 90 per group), as illustrated in Figure 1. In the intervention group, all 90 participants received the assigned nutritional intervention. In the control group, 88 participants received standard dietary advice, while two withdrew from IVF treatment before initiation. During follow-up, three participants in the intervention group discontinued the intervention (two withdrew consent and one failed to attend the follow-up visit). Three additional participants were lost to follow-up before oocyte-related outcome assessment (two withdrew consent and one failed to attend the follow-up visit). In the control group, two participants discontinued the assigned control condition (one failed to attend the follow-up visit and one started a new medication). Three additional participants were lost to follow-up before oocyte-related outcome assessment (one withdrew consent and two failed to attend follow-up visits). Overall, 167 of the 180 randomized participants (84 in the intervention group and 83 in the control group) were included in the present secondary analysis of oocyte-related outcomes. The remaining 13 participants (six in the intervention group and seven in the control group) were not included because of withdrawal from the study or unavailable outcome data. The analyses were conducted using a complete-case approach, and missing outcome data were not imputed.

Figure 1.

Consort 2025 flow diagram of the study illustrating participant enrollment, randomization, allocation to intervention and control groups, follow-up, exclusions, and inclusion in the final analysis with numbers reported at each stage.

Baseline Characteristics of the Participants

Baseline demographic and reproductive characteristics, including age, BMI, education level, occupation, duration of infertility, history of miscarriage, prior use of ART, physical activity levels, infertility etiology and AMH did not differ significantly between the two groups (Table 1).

Table 1.

General Characteristics of Women with Infertility

Variable	Intervention Group (n=84)	Control Group (n=83)	P-valuea
Age (year)	33.73± 5.76b	34.23±5.45	0.563
Weight (kg)	66.32±6.97	67.31±8.07	0.399
Height (m)	1.61±0.053	1.61±0.049	0.770
Body mass index (kg/m2)	25.43±3.19	25.87±3.53	0.396
Education level (%) Diploma or below Bachelor’s degree or higher	69% 31%	74.7% 25.3%	0.417
Job (%) Housewife Other	90.5% 9.5%	96.4% 3.6%	0.124
Supplementation intake c (% yes)	19%	10.8%	0.137
Previous pregnancy (% yes)	36.9%	24.1%	0.072
Previous delivery d (% yes)	54%	60%	0.716
Previous abortion d (% yes)	74%	65%	0.482
infertility etiology Female factor (%) Male factor (%) Both factors (%) Uncertain (%)	32.1% 17.9% 35.7% 14.3%	16.9% 28.9% 39.8% 14.5%	0.096
AMH	1.43±0.92	1.49±0.93	0.67
Duration of infertility (year)	4.62±2.91	5.42±4.03	0.145
Previous ART (% yes)	73.8%	62.7%	0.121
Type of previous ART (%) IUI IVF Both	44% 21.4% 8.3%	28.9% 24.1% 9.6%	0.215
Success rate of previous ART (n)	3.6%	3.6%	0.226
Menstrual cycle interval (day)	26.40±2.16	26.28±2.33	0.715
Menstrual cycle disorder (% yes)	13.1%	10.8%	0.654
Physical activity (MET-min/week)	1540.52±637.39	1404.80±481.14	0.123

Notes: ^aObtained from independent t-test and chi-square for continuous and categorical variables, respectively, ^bMean±SD, ^cDuring three past months, ^dAmong women with history of pregnancy.

Abbreviations: ART: Assisted reproductive technology, IVF:In vitrofertilization, IUI: Intrauterine insemination.

Dietary Intake of Micronutrients

Dietary intake was assessed at baseline (prior to the intervention) and at the end of the study. At baseline, no significant differences were observed between the intervention and control groups in energy intake, macronutrients (carbohydrate, protein, and fat), or micronutrients, including calcium, fiber, vitamin C, and vitamin B2 (P > 0.05 for all), indicating comparable dietary intake between groups before the intervention (Table 2). Following the intervention, dietary analysis revealed significantly higher intakes of fiber, calcium, vitamin C, and vitamin B2 in the intervention group compared with the control group (P < 0.001), whereas macronutrient intake, including energy, carbohydrate, protein, and fat, showed no significant differences between groups. These findings indicate good adherence to the recommended fertility-supporting dietary pattern (Table 2).

Table 2.

Dietary Intake of Selected Nutrients in Women with Infertility During the Study

Nutrients		Intervention Group (n=84)	Control Group (n=83)	P1-valuea
Energy (kcal)	Baseline End P2c	2053.67±298.54b 2054.39±227.68 0.975	2005.56±199.69 2000.14±219.57 0.725	0.223 0.119
Carbohydrate (g)	Baseline End P2	270.79±49.66 271.33±34.18 0.888	266.38±32.30 265.04±31.95 0.638	0.684 0.855
Protein (g)	Baseline End P2	77.78±21.14 81.00±16.20 0.168	76.24±18.75 77.21±16.68 0.424	0.715 0.519
Fat (g)	Baseline End P2	73.55±19.77 71.67±12.00 0.379	70.56±9.59 70.13±11.03 0.489	0.478 0.781
Calcium (mg)	Baseline End P2	647.93±211.89 999.53±182.17 <0.001	711.75±241.34 713.81±235.07 0.461	0.073 <0.001
Fiber (g)	Baseline End P2	15.24±4.11 27.05±4.77 <0.001	16.10±4.79 16.20±4.75 0.145	0.275 <0.001
Vitamin C (mg)	Baseline End P2	100.78±32.53 200.49±62.98 <0.001	94.16±19.44 94.83±23.47 0.166	0.126 <0.001
Vitamin B2 (mg)	Baseline End P2	0.89±0.31 1.17±0.41 <0.001	0.97±0.29 0.94±0.27 0.119	0.092 <0.001

Notes: ^aObtained from independent t-test for energy and ANCOVA for other nutrients (adjusted for energy intake), ^b Mean±SD, c Obtained from paired sample t-test.

Biochemical Micronutrient Status

As reported in the Table 3, at the end of the 8-week intervention period, serum levels of folate, vitamin B12, and 25-hydroxyvitamin D significantly increased in the intervention group compared to baseline (P<0.001 for all). In the control group, although participants received 500 micrograms of folic acid as part of standard preconception care, only serum folate showed a slight but statistically significant increase (P<0.001), while changes in vitamin B12 (P = 0.849) and 25(OH)D (P = 0.095) levels were not significant. Between-group comparisons at the end of the study, adjusted for baseline values using ANCOVA, indicated that serum concentrations of all three micronutrients were significantly higher in the intervention group than in the control group (P < 0.001 for all).

Table 3.

Serum Concentration of Folate, Vitamin B12 and 25-Hydroxyvitamin D in Women with Infertility at Baseline and After 8 weeks of Intervention

Variables	Intervention Group (n=84)			Control Group (n=83)			P-valuec
	Baseline	End	P-valueb	Baseline	End	P-valueb
Folate (ng/mL)	17.39±8.10a	29.59±8.36	<0.001	15.07±7.16	17.52±6.16	<0.001	<0.001
Vitamin B12 (pg/mL)	359.01±99.23	466.18±66.29	<0.001	379.40±158.15	378.87±156.89	0.849	<0.001
25-hydroxyvitamin D (ng/mL)	31.83±11.85	38.90±9.94	<0.001	32.33±11.24	31.79±11.73	0.095	<0.001

Notes: ^a Mean±SD. ^b Obtained from paired t-test comparing baseline and end of trial values within each group. ^c Obtained from ANCOVA comparing end of trial values between two groups adjusted for baseline.

Oocyte-Related Outcomes

Oocyte-related outcomes following the 8-week intervention are presented in Table 4. After adjustment for baseline age and BMI using Poisson regression with robust standard errors, no statistically significant differences were observed between the intervention and control groups in AFC (7.48 ± 3.55 vs 7.23 ± 3.12; IRR = 1.02, 95% CI: 0.90–1.14; P = 0.806) or COC count (6.24 ± 3.33 vs 6.19 ± 3.43; IRR = 0.99, 95% CI: 0.85–1.14; P = 0.858). In contrast, the intervention group had significantly higher numbers of MII oocytes (5.76 ± 2.98 vs 4.89 ± 2.40; IRR = 1.16, 95% CI: 1.01–1.33; P = 0.035) and 2PN zygotes (4.71 ± 2.70 vs 3.37 ± 1.92; IRR = 1.38, 95% CI: 1.17–1.61; P < 0.001) compared with the control group, corresponding to 16% and 38% higher expected counts, respectively. To account for multiple testing across the four newly reported secondary outcomes (AFC, COC, MII, and 2PN), sensitivity analyses were performed using the Holm and Benjamini–Hochberg (BH) procedures (Table S1). After correction for multiple comparisons, the higher 2PN zygote count in the intervention group remained statistically significant (adjusted P < 0.001 with both methods). However, the difference in MII oocyte count, which was statistically significant in the nominal analysis (P = 0.035), was no longer statistically significant after Holm (P = 0.104) or BH correction (P = 0.069). No statistically significant differences were observed for AFC or COC counts after either correction.

Table 4.

Oocyte-Related Outcomes in Women with Infertility After the 8-Week Intervention

Outcome	Intervention (n=84) Mean ± SD	Intervention Median (Q1–Q3)	Control (n=83) Mean ± SD	Control Median (Q1–Q3)	Adjusted effect (95% CI)a	P-valuea
AFC (n)	7.48±3.55	7.00 (4–10)	7.23±3.12	7.00 (4–10)	IRR 1.02 (0.90–1.14)	0.806
COCs (n)	6.24±3.33	6.00 (4–8)	6.19±3.43	6.00 (3–9)	IRR 0.99 (0.85–1.14)	0.858
MII oocytes (n)	5.76±2.98	5.00 (3.25–8)	4.89±2.40	5.00 (3–7)	IRR 1.16 (1.01–1.33)	0.035
2PN zygotes (n)	4.71±2.70	4.00 (3–7)	3.37±1.92	3.00 (2–5)	IRR 1.38 (1.17–1.61)	<0.001
Fertilization rateb	0.81±0.16	0.82 (0.75–0.90)	0.66±0.24	0.71 (0.50–0.83)	OR 1.92 (1.41–2.63)	<0.001

Notes: Fertilization rate was previously reported from the same randomized controlled trial¹³ and is included here for contextual interpretation rather than as a novel outcome. ^a Count outcomes: Poisson regression with robust (sandwich) standard errors, adjusted for age and BMI; effect is the incidence rate ratio (IRR; control = reference). Fertilization rate: binomial (logistic) regression of the number of 2PN zygotes out of the number of MII oocytes, with robust standard errors, adjusted for age and BMI; effect is the odds ratio (OR). Model-based adjusted fertilization proportions were 0.81 (intervention) and 0.66 (control). ^b Number of 2PN zygotes divided by number of MII oocytes (participant-specific denominator). Regression analysis included 166 women (one record with 2PN > MII was excluded).

Abreviations: AFC, antral follicle count; COC, cumulus–oocyte complex; MII, metaphase II; 2PN, two-pronuclear; IRR, incidence rate ratio; OR, odds ratio.

Furthermore, the fertilization rate was higher in the intervention group than in the control group (0.81 ± 0.16 vs 0.66 ± 0.24). Adjusted binomial logistic regression showed significantly higher odds of fertilization in the intervention group (OR = 1.92, 95% CI: 1.41–2.63; P < 0.001). This analysis included 166 participants. As previously reported from the same randomized controlled trial,¹³ the fertilization rate is presented here to provide context for the newly reported oocyte-related outcomes rather than as a novel finding.

Discussion

The present secondary analysis of a randomized controlled trial evaluated the effects of a PFD combined with omega-3 supplementation on oocyte-related outcomes in women undergoing IVF/ICSI. Compared with the control group, the intervention group had significantly higher numbers MII oocytes and 2PN zygotes, whereas no statistically significant differences were observed in AFC or COC count. A higher fertilization rate was also observed, as previously reported from the same trial.¹³ These findings suggest potential benefits of the combined nutritional intervention for specific oocyte-related laboratory outcomes; however, the independent contributions of individual dietary components and supplements cannot be determined.

Importantly, although the higher MII oocyte count was statistically significant in the initial adjusted Poisson regression analysis, this finding did not remain statistically significant after correction for multiple comparisons using either the Holm or Benjamini–Hochberg (BH) procedure. In contrast, the higher 2PN zygote count remained statistically significant after both corrections. Thus, the evidence for a difference in 2PN zygote count was more robust to multiplicity adjustment than that for MII oocyte count. Given the exploratory nature of these secondary analyses, particularly the sensitivity of the MII finding to multiplicity adjustment, the results should be interpreted cautiously and confirmed in future adequately powered randomized controlled trials.

The PFD was originally developed from observational evidence linking combinations of dietary factors and nutrient supplementation with reproductive outcomes.²⁰ At the time the parent trial was designed, interventional evidence evaluating this dietary pattern as a whole was limited. The intervention was therefore designed to assess a comprehensive nutritional approach, reflecting the fact that dietary modifications and supplements may be used together in clinical practice rather than in isolation. Because the intervention was evaluated as a package, previous studies of individual foods and nutrients are discussed below to provide biological context and identify plausible mechanisms, not to establish that any specific component caused the observed differences.

Oocyte maturation and normal fertilization are important intermediate laboratory outcomes during IVF, and nutritional factors have been investigated in relation to reproductive health.²¹ However, the present secondary analysis did not assess clinical pregnancy, live birth, the number of repeated IVF cycles, or healthcare costs. Although repeated IVF treatment can impose psychological and financial burdens,²² it cannot be inferred from the present results that this intervention reduces treatment burden, improves overall IVF success, or is cost-effective. The clinical significance of the observed laboratory differences therefore requires confirmation using patient-important reproductive outcomes.

Folate and vitamin B12 are among the nutrients included in the combined intervention, but the higher MII oocyte and 2PN zygote counts cannot be attributed specifically to either supplement. In a retrospective study, Cirillo et al (2021) reported higher mean numbers of MII oocytes and higher 2PN fertilization rates among women using a supplement containing 5-methyltetrahydrofolate, vitamin B12, and vitamin B6 than among women using folic acid alone (P = 0.04 and P = 0.05, respectively).²³ Vitamin B12 plays a central role in one-carbon metabolism and is essential, along with folate, for DNA synthesis and methylation. Disruption of one-carbon metabolism and elevated homocysteine concentrations have been proposed as pathways relevant to oocyte maturation and reproductive outcomes.²⁴ In our trial, serum folate and vitamin B12 concentrations increased in the intervention group, but these biochemical changes do not demonstrate that either nutrient mediated the observed differences in oocyte-related outcomes.

The higher fertilization rate following the eight-week intervention, previously reported from this trial,¹³ is also consistent with earlier research investigating omega-3 sources. For example, Chu et al (2024) reported higher MII oocyte and fertilization rates following flaxseed oil supplementation among women with decreased ovarian reserve undergoing IVF.²⁵ Omega-3 fatty acids have been investigated for their possible roles in membrane lipid composition, lipid metabolism, and cellular signaling pathways relevant to oocyte development.^10,25 Experimental mechanisms proposed in the literature also include changes in granulosa-cell signaling and ovarian steroidogenesis; however, these pathways were not measured in the present study. Furthermore, the present intervention combined marine omega-3 supplementation with multiple dietary changes and other supplements, preventing attribution of the observed effects specifically to omega-3 fatty acids.

Vitamin D was one of the components of our intervention. Vitamin D signaling occurs in reproductive tissues and has been implicated in ovarian steroidogenesis, follicular development, and the regulation of anti-Müllerian hormone (AMH).²⁶ Evidence from experimental studies suggests that vitamin D status may influence follicular development and gonadotropin-related pathways, although the relevance of these mechanisms to clinical IVF outcomes remains uncertain.²⁶ Liu et al (2019) reported that lower serum vitamin D levels were associated with reduced fertilization rates, while higher vitamin D levels correlated with improved fertilization outcomes.²⁷ Although serum 25-hydroxyvitamin D concentrations increased in our intervention group, the present design cannot establish whether this change contributed to the differences in MII or 2PN counts.

Whole grains are another important component of the PFD and may be relevant to reproductive health through metabolic and inflammation-related pathways. In a prospective observational study of healthy premenopausal women, greater whole-grain intake was associated with lower serum high-sensitivity C-reactive protein concentrations.²⁸ Lignans present in whole grains have also been investigated for their potential interactions with estrogen-related pathways.²⁹ These findings offer possible biological explanations for the inclusion of whole grains in the PFD, but they do not establish an effect of whole grains on oocyte maturation in the present trial. Inflammatory markers, insulin resistance, and lignan exposure were not assessed as mediators of the observed outcomes.

Soy and its isoflavones are key components of the PFD.²⁰ In an observational study of women undergoing ART, soy food intake was associated with more favorable reproductive treatment outcomes.³⁰ Potential explanations include the estrogen receptor activity and antioxidant properties of soy isoflavones, although these mechanisms were not directly evaluated in our trial.³⁰ In addition, the PFD recommends reducing the use of disposable plastic food containers, partly to limit potential exposure to endocrine-disrupting compounds such as bisphenol A (BPA).²⁰ Experimental and human evidence has raised concerns about associations between BPA exposure and oocyte health, including pathways involving oxidative stress, estrogen-receptor signaling, meiotic spindle integrity, and epigenetic changes.³¹ However, BPA exposure was not measured in the present study, and neither a protective effect of soy nor an effect mediated by reduced BPA exposure can be inferred from our findings.

Fruit and vegetable selection is also emphasized in the PFD, including a preference for produce categorized as having lower pesticide residues.⁷ In a prospective observational study, Chiu et al reported that higher intake of fruits and vegetables classified as having high pesticide residues was associated with lower probabilities of clinical pregnancy and live birth among women undergoing ART; intake of low-residue produce was not significantly associated with these outcomes.³² These observations provide a rationale for discussing potential dietary pesticide exposure in the context of fertility-focused nutrition, but they do not establish a causal effect on oocyte maturation. Because pesticide exposure and endocrine-disrupting chemicals were not quantified in our trial, any contribution of reduced exposure to the observed oocyte-related outcomes remains hypothetical.

Taken together, previous studies of individual nutrients and foods suggest several plausible pathways through which a comprehensive nutritional intervention might influence reproductive biology. Nevertheless, the present study assessed the combined intervention and did not test the separate effects of its components, possible interactions among them, or the proposed biological mechanisms. The prespecified oocyte-related outcomes were analyzed as secondary outcomes of a trial whose sample size was determined by pregnancy rate; therefore, limited statistical power for smaller effects and the possibility of chance findings across multiple outcomes should also be considered. Future adequately powered randomized trials could evaluate the overall intervention in relation to clinical pregnancy and live birth and, where feasible, use designs that distinguish the contributions of dietary components and supplements. Until such evidence is available, the present results should be interpreted as exploratory evidence concerning MII oocyte and 2PN zygote counts, rather than proof of the efficacy of specific nutrients or of broader clinical benefits.

Several limitations of the present study should be acknowledged. First, although the gynecologist and embryologists responsible for outcome assessment were blinded to treatment allocation, participants could not be blinded to the dietary intervention, potentially introducing performance bias. However, the use of objective laboratory and ultrasonographic outcomes may have reduced the risk of detection bias. Furthermore, the multi-component nature of the intervention, which combined a PFD with omega-3 supplementation, prevented the identification of the independent contributions of individual components or their potential interactions. Therefore, the observed findings should be interpreted in relation to the combined nutritional intervention rather than attributed to specific nutrients or dietary components.

Another limitation relates to dietary assessment. Dietary intake was assessed using three-day dietary records collected at baseline and at the end of the intervention, which may not fully reflect habitual dietary intake and may be subject to reporting errors. The inclusion of a validated food frequency questionnaire (FFQ) could have provided complementary information on usual dietary patterns. Additionally, anthropometric assessment did not include waist circumference, limiting a more comprehensive evaluation of body fat distribution.

An additional limitation was the lack of detailed information on certain ART-related clinical factors, including ovarian stimulation protocols, gonadotropin doses, and ovulation-triggering regimens. Consequently, potential differences in these factors between the study groups could not be assessed or accounted for in the statistical analyses. Although randomization was intended to balance prognostic factors between groups, imbalances in these unmeasured treatment-related characteristics cannot be excluded.

Another limitation concerns the analysis population. The analyses were not performed according to the intention-to-treat principle: of the 180 randomized women, 13 (6 in the intervention group and 7 in the control group) were excluded because of withdrawal, discontinuation or unavailable outcome data, and the 167 women with available data were analyzed (per-protocol/complete-case analysis) without imputation of missing outcomes. Although attrition was modest (7.2%) and similar between groups, excluding randomized participants may break the balance achieved by randomization and introduce attrition bias, so the effect estimates may differ from those of an ITT analysis. In addition, because several secondary outcomes were tested, the results should be regarded as exploratory; after correction for multiple comparisons (Holm and Benjamini–Hochberg), the difference in 2PN zygotes remained significant, whereas the difference in MII oocytes did not.

Finally, the sample size was calculated based on pregnancy rate as the primary outcome of the parent randomized controlled trial rather than the oocyte-related secondary outcomes investigated in the present analysis. Although these outcomes were planned as part of the original trial, the sample size was not specifically determined to ensure adequate statistical power for each secondary outcome. Consequently, the possibility of insufficient statistical power, particularly for detecting smaller effects, cannot be excluded. Moreover, the assessment of multiple secondary outcomes raises the possibility of chance findings. Accordingly, the results should be interpreted cautiously as exploratory findings and require confirmation in future adequately powered randomized controlled trials. Although clinical pregnancy outcomes were evaluated in a separate analysis of the parent randomized controlled trial, the present analysis did not specifically examine the relationship between oocyte-related laboratory outcomes and subsequent clinical pregnancy. Therefore, the clinical implications of the observed differences in MII oocyte and 2PN zygote counts remain to be established.

Strengths of this study include the use of objective biological markers—serum folate, vitamin B12, and vitamin D concentrations—to assess adherence to the dietary and supplementation intervention, thereby reducing reliance on self-reported intake and minimizing reporting bias. The intervention itself was designed as a comprehensive, evidence-based PFD combined with targeted omega-3 supplementation, representing an integrated nutritional approach to improving reproductive outcomes. Furthermore, major confounding variables, including age and BMI, were controlled for in the analyses, strengthening the internal validity of the findings.

Conclusion

In this exploratory secondary analysis of a randomized controlled trial, a PFD combined with omega-3 supplementation was associated with a higher number of 2PN zygotes among women undergoing IVF/ICSI, a finding that remained statistically significant after correction for multiple comparisons. Although the intervention group also had a higher number MII oocytes in the initial adjusted analysis, this finding did not remain statistically significant after multiplicity adjustment. No statistically significant between-group differences were observed in AFC or COC count. These findings suggest a potential benefit of the combined nutritional intervention for certain fertilization-related outcomes. However, given the exploratory nature of the secondary analyses, the results should be interpreted cautiously, and further adequately powered randomized controlled trials are needed to confirm these findings and determine their clinical significance.

Funding Statement

This research was financially supported by Isfahan University of Medical Sciences, Isfahan, Iran.

Declaration of Generative AI in Scientific Writing

This technology was only used to improve readability and language of the manuscript. It was with human oversight and control, and authors carefully review and edit the result. AI tools was not used to analyse and draw insights from data as part of the research process.

Trial Registration

Iranian Registry of Clinical Trials (IRCT20130903014551N13, https://irct.ir/trial/71704)

Registration number: IRCT20130903014551N13

Registry URL: https://irct.ir/trial/71704

Date of registration: 12 August 2023

Date of enrollment of the first participant:4 June 2024

Abbreviations

AFC, Antral Follicle Count; AMH, Anti-Müllerian Hormone; ART, Assisted Reproductive Technology; BMI, Body Mass Index; COC, Cumulus–Oocyte Complex; IVF, In Vitro Fertilization; MII, Metaphase II; PFD, Pro-Fertility Diet; 2PN, Two-Pronuclear Zygote.

Data Sharing Statement

Data will be available on request. Please contact to following email: sm_rouhani@nutr.mui.ac.ir

Ethics Approval and Consent to Participate

The study was conducted in accordance with the ethical principles of the Declaration of Helsinki (2013 revision). Ethical approval was obtained from the Ethics Committee of Isfahan University of Medical Sciences (approval code: IR.ARI.MUI.REC.1402.091). The trial was prospectively registered in the Iranian Registry of Clinical Trials (IRCT20130903014551N13). Written informed consent was obtained from all participants prior to enrollment.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.