Reverse Effects of Nicotinamide Mononucleotide Supplementation on Declining Quality of Oocytes With Polycystic Ovary Syndrome

ABSTRACT

Polycystic ovary syndrome (PCOS) adversely impacts oocyte quality, underscoring effective interventions as a central focus of research. Our study demonstrates that nicotinamide mononucleotide (NMN) not only restores NAD⁺ homeostasis in oocytes from PCOS mice but also enhances the developmental rate of PCOS oocytes during in vitro maturation (IVM). Transcriptomic analysis and experimental validation suggest that NMN helps reverse the decline in PCOS oocyte quality by mitigating oxidative stress, improving mitochondrial function, and preserving spindle morphology. Mechanistically, we found that NMN supplementation upregulates SIRT1 expression, which responds to fluctuations in NAD⁺ levels through the NAD⁺ salvage pathway regulated by nicotinamide mononucleotide adenylyltransferase (NMNAT). In conclusion, our findings highlight the potential of NMN in improving PCOS oocyte quality, offering valuable insights for clinical PCOS treatment and the advancement of assisted reproductive technology.

Supplementation with NMN improves oocyte quality in a PCOS mouse model by restoring NAD⁺ levels, reducing oxidative stress, enhancing mitochondrial function, and preserving spindle integrity. Mechanistically, NMN activates SIRT1 expression via the NAD⁺ salvage pathway, offering a promising strategy to enhance in vitro maturation and fertility outcomes.

1. Introduction

Polycystic ovary syndrome (PCOS) is a prevalent and complex endocrine disorder characterized by hyperandrogenism, disrupted ovulation, and polycystic ovarian morphology [1]. Although assisted reproductive technology (ART) has successfully facilitated childbirth in patients with PCOS, it is essential to recognize the detrimental effects of PCOS and its associated complications on oocytes. These include elevated levels of reactive oxygen species (ROS) [2], chronic inflammation [3], neuroendocrine alterations [4], and insulin resistance [5]. Clinical studies have identified a significant correlation between an abnormal ovarian microenvironment and reduced oocyte quality in PCOS patients [6], leading to lower fertilization rates and compromised embryonic developmental potential [7, 8].

To enhance egg quality and improve pregnancy outcomes in PCOS patients, various antioxidants are used in vivo and in vitro to help PCOS oocytes better withstand stress during ART [9, 10, 11]. Nicotinamide adenine dinucleotide, oxidized form (NAD⁺) is a crucial redox cofactor involved in multiple biochemical processes [12, 13, 14]. Studies have reported a significant reduction in NAD⁺ levels in granulosa cells of PCOS patients and the muscle tissue of PCOS mice, leading to increased inflammatory factors and insulin resistance [15, 16]. Restoring NAD⁺ homeostasis may serve as an effective intervention in PCOS models.

In mammals, NAD⁺ biosynthesis occurs via three pathways: de novo synthesis from tryptophan, the Preiss‐Handler pathway from nicotinic acid, and the salvage pathway utilizing NAD⁺ precursors [17]. Certain NAD⁺ precursors, such as N1‐methylnicotinamide, nicotinamide, and nicotinamide riboside, have shown efficacy in mitigating the hyperandrogenic state and mitochondrial dysfunction associated with PCOS [15, 18]. As a key intermediate in the salvage pathway, regulated by nicotinamide mononucleotide adenylyltransferase (NMNAT), nicotinamide mononucleotide (NMN) has demonstrated therapeutic potential in various conditions, including aging, diabetes, obesity, and cardiovascular diseases [19, 20]. While NMN supplementation has been shown to enhance muscle insulin sensitivity in PCOS mice, its potential effects on oocyte quality in PCOS remain to be explored.

In order to benefit PCOS patients from ART, we utilized a mouse model with PCOS and found that decreased NAD⁺ levels and low developmental potential in PCOS oocytes were elevated by the addition of NMN in vitro. The transcriptional sequencing results suggested a key factor of the NAD⁺/SIRT1 axis contributing to the quality of PCOS oocytes, and elevated oxidative stress, mitochondrial dysfunction, spindle abnormality, and apoptotic signal were all alleviated in PCOS oocytes after NMN supplementation. We further showed that the metabolism of supplemented NMN in oocytes was regulated by NAMPTs in the salvage pathway, which affected the expression of Sirt1. This study provides a certain experimental basis for the improvement of oocyte quality and IVM success rate in PCOS patients in clinical practice.

2. Materials and Methods

2.1. Mice

ICR female mice (3 weeks old) were used in this study. The mice were housed under a 12‐h light/dark cycle and allowed to acclimate for 3 days before the experiment. After acclimatization, they were randomly assigned to either the control group or the PCOS group. To induce the PCOS model, mice in the PCOS group received daily subcutaneous injections of dehydroepiandrosterone (DHEA; 6 mg/100 g body weight; MedChemExpress, USA) dissolved in sesame oil (YuanYe, China) for 21 consecutive days. Mice in the control group were administered an equal volume of sesame oil. Beginning on day 11 of treatment, daily vaginal smears were performed for 10 consecutive days to monitor the estrous cycle and confirm the successful establishment of the PCOS model. Mouse experiments were carried out according to and with the approval of the Institutional Animal Care and Use Committee (Nanjing Medical University). Animal experiments followed the Guide for the Care and Use of Laboratory Animals.

2.2. Determination of Serum Hormones

After the final day of intervention, the mice were fasted from 8:00 PM onward and anesthetized at 8:00 AM the following morning via intraperitoneal injection of a 10% chloral hydrate solution (Sigma‐Aldrich, USA). Blood samples were collected to obtain serum, and the concentrations of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), and testosterone were measured using enzyme‐linked immunosorbent assay (ELISA) kits (Jiangsu Meimian Industrial Company Co. Ltd., China). All procedures were performed strictly according to the manufacturer's instructions.

2.3. Histological Analysis of Ovaries

For histological analysis, ovaries were collected from each group of mice and fixed in 4% paraformaldehyde (PFA) at 4°C overnight. Following fixation, the ovaries were dehydrated, embedded in paraffin, and sectioned into 5‐μm‐thick slices for hematoxylin and eosin (H&E) staining.

2.4. Oocyte Collection and Culture

Oocytes were isolated from female mice in the PCOS and control groups. To obtain fully grown germinal vesicle (GV) oocytes, mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG). After 48 h, cumulus‐oocyte complexes were retrieved by manually rupturing the antral ovarian follicles, and cumulus cells were removed through repeated mouth‐pipetting. For in vitro maturation (IVM), GV oocytes were cultured in IVM medium (Nanjing Aibei Biotechnology Co. Ltd., China) at 37°C under 5% CO₂. The germinal vesicle breakdown (GVBD) rate and first polar body (PB1) extrusion rate were assessed after 3 and 15 h of culture, respectively. To investigate the effects of nicotinamide mononucleotide (β‐NMN), different concentrations (0, 0.1, 1, 10, and 100 μM; Sigma‐Aldrich) were added to the IVM medium of PCOS mouse oocytes.

2.5. Measurement of NAD ⁺ Levels

NAD⁺ levels were measured using a commercial assay kit (Abcam, USA) following the manufacturer's protocol. A total of 160 oocytes were collected for NAD⁺ extraction and quantification, following the procedure described by Xinghan Wu et al. [21]. Briefly, total NAD (NAD⁺ and NADH) and NADH levels were quantified using a colorimetric assay at 450 nm with the SPARK CYT4000 multi‐functional microplate reader. The NAD⁺ concentration was calculated by subtracting the NADH value from the total NAD.

2.6. Immunofluorescence

Oocytes were fixed in 4% paraformaldehyde (PFA) for 30 min and then permeabilized with 0.5% Triton X‐100 for 20 min. After blocking with PBS containing 1% bovine serum albumin (BSA) for 1 h, samples were incubated overnight at 4°C with primary antibodies. To visualize the spindle, oocytes were stained with an α‐tubulin monoclonal antibody (Thermo Fisher Scientific, USA). Chromosomes were labeled with Hoechst 33342 (Thermo Fisher Scientific) for 10 min. Following PBS washes, oocytes were mounted in anti‐fade medium (Beyotime Biotechnology, China) and examined using a laser scanning confocal microscope (Zeiss, Germany).

To assess reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), glutathione (GSH) levels, ATP levels, and apoptosis, several fluorescent probes were used. The probes included dichlorodihydrofluorescein diacetate (Beyotime Biotechnology) for ROS, 500 nM cell‐permeant MitoTracker Red CMXRos (Thermo Fisher Scientific) for mitochondria, 10 μmol/L monochlorobimane (MedChemExpress) for GSH, ATP‐Red 1 (MedChemExpress) for ATP, an enhanced MMP assay kit with JC‐1 (Beyotime Biotechnology) for mitochondrial membrane potential, and Annexin V probes (Beyotime Biotechnology) for apoptosis. Briefly, oocytes were incubated in M2 medium containing the respective fluorescent probe for 30 min at 37°C in a dark environment with 5% CO₂. After incubation, oocytes were washed five times with PBS and immediately analyzed using a laser scanning confocal microscope.

2.7. Plasmid Construction and mRNA Synthesis

The pcDNA3.1(+)‐Sirt1‐Flag plasmid was purchased from General Biology. mRNA was synthesized via in vitro transcription using the HiScribe T7 ARCA mRNA Kit (New England Biolabs, USA), following the manufacturer's instructions. The synthesized RNA was aliquoted and stored at −80°C until further use.

2.8. SIRT1, NMNAT1, NMNAT2, NMNAT3 Knockdown and SIRT1 Overexpression

Small interfering RNAs (siRNAs) targeting Sirt1, Nmnat1, Nmnat2, and Nmnat3 were purchased from General Biol (China) and diluted in RNase‐free water to a final concentration of 20 μmol/L. A total of 2.5 μL of the siRNA solution was microinjected into oocytes. For overexpression experiments, 10 μL of capped mRNA solution (500 ng/μL) was microinjected into oocytes. Following injection, oocytes were arrested at the GV stage in medium containing 2.5 μmol/L milrinone (MedChemExpress) for 20 h to allow mRNA degradation or translation. The relevant primer sequences are provided in Table S5.

2.9. Quantitative Real‐Time PCR

A total of 30 oocytes were collected for RNA extraction using the Dynabeads mRNA DIRECT Kit (Thermo Fisher Scientific), followed by complementary DNA (cDNA) synthesis with the HiScript II RT SuperMix (Vazyme, China). Quantitative real‐time PCR (RT‐qPCR) was performed using ChamQ SYBR qPCR Master Mix (Vazyme) on a QuantStudio 7 Flex Real‐Time PCR System (Thermo Fisher Scientific). Relative mRNA levels were normalized to 18S rRNA, and fold changes were calculated using the comparative Ct method. Primers were synthesized by General Biol, and their sequences are listed in Table S5.

2.10. Immunoblotting

Oocytes collected from each group were lysed, and proteins were separated by SDS‐PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in Tris‐buffered saline (TBS) containing 0.1% Tween 20 and 5% low‐fat milk for 1 h, followed by overnight incubation at 4°C with primary antibodies, including rabbit anti‐SIRT1 antibody (Cell Signaling Technology, CST, USA) and anti‐FLAG antibody (Abclonal, China).

After multiple washes with TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence (ECL) Western blot detection system. The membranes were then washed and reprobed with an anti‐β‐actin antibody (Abclonal) as a loading control.

2.11. RNA Sequencing and Analysis

Three sets of in vitro matured oocytes were collected from each group (15 oocytes per sample). RNA sequencing libraries were generated using the Smart‐seq2 protocol [22]. Briefly, samples were lysed, and reverse transcription was performed using Oligo(dT) primers (Thermo Fisher) to synthesize first‐strand complementary DNA (cDNA). The library was then constructed through DNA fragmentation, end repair, and PCR amplification. Sequencing was performed on the Illumina NovaSeq 6000 platform (LC‐Bio Technology Co. Ltd., China) with 150‐bp paired‐end reads.

Raw sequencing reads were processed using Cutadapt to remove low‐quality sequences and adapter contamination, resulting in high‐quality clean reads. Sequence quality was then assessed with FastQC. Transcript expression levels were quantified using StringTie and Ballgown, and mRNA abundance was measured by calculating fragments per kilobase of transcript per million mapped reads (FPKM) values. Differential expression analysis was conducted using the R package DESeq2. Genes with an adjusted p‐value < 0.05 and an absolute fold change > 2 were identified as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using DAVID.

2.12. Statistical Analysis

All data were analyzed using GraphPad Prism 10 statistical software. Measurement data were tested for normality and expressed as the mean ± standard error of the mean (SEM). An unpaired two‐tailed t‐test was used for comparisons between two groups, while one‐way analysis of variance (ANOVA) was applied for comparisons among multiple groups. Statistical significance was defined as follows: N.S., not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001.

3. Results

3.1. In Vitro Supplementation With NMN Enhances the Development of PCOS Oocytes

A PCOS mouse model was established by continuous subcutaneous injection of dehydroepiandrosterone for 21 days, followed by administration of PMSG to obtain germinal vesicle (GV) oocytes (Figure 1A). To confirm the successful establishment of the model, a series of assessments, including estrous cycle analysis, hormone level measurements, and ovarian histology, were conducted, all of which validated the induction of PCOS in mice (Figure S1A–G). Next, GV oocytes derived from PCOS mice were cultured in vitro. The results demonstrated more than 20% reduced rates of germinal vesicle breakdown (GVBD) and first polar body (PB1) extrusion in PCOS oocytes compared to normal and control groups, which exhibited similar developmental rates (Figure S1H,I). Further analysis revealed a 67% reduction in NAD⁺ levels in PCOS GV oocytes (Figure S1J), suggesting that supplementation with NAD⁺ precursors could counteract impaired oocyte development by restoring NAD⁺ homeostasis. As a key intermediate in NAD⁺ biosynthesis, NMN was tested for its ability to enhance PCOS oocyte development in a concentration‐gradient experiment (0, 0.1, 1, 10, and 100 μmol/L) that was according to the concentration range in previous studies [23, 24]. Among these, 1 μmol/L NMN exhibited the most pronounced effect (Figure 1B–D) and was therefore selected for subsequent experiments. Notably, NMN supplementation significantly increased NAD⁺ levels in PCOS MII oocytes (Figure 1E). In conclusion, GV oocytes derived from PCOS mice exhibit impaired maturation in vitro, and appropriate NMN supplementation enhances their developmental potential.

FIGURE 1.

NMN enhances the development of PCOS oocytes during the IVM procedure. (A) Schematic diagram illustrating the establishment of the PCOS mouse model and the application of NMN during IVM. (B) Representative images of PCOS oocytes cultured in vitro under different NMN concentrations. Scale bar = 100 μm. (C, D) Quantitative analysis of GVBD rate (C) and PB1 extrusion rate (D) in PCOS oocytes treated with varying NMN concentrations. A total of 60 oocytes were analyzed per group. (E) NAD⁺ levels measured in control, PCOS, and PCOS+NMN MII oocytes. A total of 80 oocytes were collected per group. Graphs represent mean ± SEM from three independent experiments. Statistical significance was determined using an unpaired two‐tailed t‐test: ns = not significant, *p < 0.05, **p < 0.01.

3.2. NMN Supplementation Reverses the Inferior Quality of PCOS Oocytes

To investigate the mechanisms underlying the improved developmental potential of PCOS oocytes following NMN supplementation, metaphase II (MII) oocytes matured in vitro were collected from the control, PCOS, and PCOS with NMN supplementation (PCOS+NMN) groups for transcriptome profiling. Three replicates were performed for each group, with 15–20 oocytes collected per sample. The number of raw and mapped reads was comparable across the three groups (Figure S2A,B), and no significant differences were observed in the number of detected genes using a fragments per kilobase million (FPKM) threshold of > 1 (Figure S2C). Differential expression gene (DEG) analysis (fold change > 2, adjusted p‐value < 0.05, Table S1) revealed 45 upregulated and 127 downregulated DEGs in PCOS oocytes compared to the control group (Figure 2A,B). Additionally, 154 upregulated and 368 downregulated DEGs were identified when comparing PCOS to PCOS+NMN oocytes (Figure 2A,C), while 93 upregulated and 322 downregulated DEGs were detected between PCOS+NMN and the control group (Figure 2A; Figure S2D). Gene Ontology (GO) analysis indicated that biological processes related to transcription, cell cycle regulation, apoptosis, and oxidative stress response were significantly enriched in DEGs from both the control versus PCOS and PCOS versus PCOS+NMN comparisons (Figure 2D,E, Table S2). Similarly, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted overlapping enrichment in RNA degradation, hormone signaling pathways, and mitophagy (Figure 2F,G, Table S2). Collectively, these findings suggest that NMN supplementation restores the transcriptional profile of PCOS oocytes.

FIGURE 2.

Effect of NMN on the transcriptome on PCOS oocytes. (A) Heatmap showing differentially expressed genes (DEGs) (fold change > 2, adjusted p‐value < 0.05) in control, PCOS, and PCOS+NMN oocytes. (B, C) Volcano plots illustrating upregulated and downregulated DEGs in control vs. PCOS (B) and PCOS vs. PCOS+NMN (C). (D, E) Gene Ontology (GO) analysis of biological processes associated with DEGs in control vs. PCOS (D) and PCOS vs. PCOS+NMN (E). (F, G) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in control vs. PCOS (F) and PCOS vs. PCOS+NMN (G).

Based on the enriched pathways and biological processes of DEGs, we performed further functional validation in PCOS oocytes. First, we observed significantly higher reactive oxygen species (ROS) levels in PCOS oocytes compared to control oocytes, while NMN supplementation effectively reduced ROS accumulation (Figure 3A,B). Additionally, glutathione (GSH) content, a key indicator of cellular antioxidant capacity [25], was markedly decreased in PCOS oocytes but partially restored following NMN treatment (Figure S3A,B). Next, we examined mitochondrial function. Fluorescence imaging revealed that mitochondria were predominantly distributed around the chromosomes in control oocytes, whereas in PCOS oocytes, they exhibited clustered aggregation within the cytoplasm (Figure 3C). NMN treatment significantly reduced the proportion of oocytes exhibiting abnormal mitochondrial distribution (Figure 3D). Moreover, NMN supplementation effectively restored mitochondrial membrane potential (MMP) and increased ATP content in PCOS oocytes (Figure S3C–F). Spindle morphology, a critical indicator of oocyte developmental competence, was significantly disrupted in the PCOS group (Figure 3E). However, NMN supplementation reduced the spindle abnormality rate from approximately 46% to 24%, bringing it closer to normal levels (Figure 3F). In conclusion, NMN supplementation partially mitigates PCOS‐induced oocyte deterioration through multiple pathways, including oxidative stress reduction, mitochondrial function restoration, and spindle structure stabilization.

FIGURE 3.

Enhancement of NMN on biological processes in PCOS oocytes. (A) Representative images showing reactive oxygen species (ROS) levels in control, PCOS, and PCOS+NMN oocytes. Scale bar = 20 μm. (B) Quantification of ROS levels among the three groups. A total of 20 oocytes were analyzed per group. (C) Mitochondrial distribution in control, PCOS, and PCOS+NMN oocytes stained with MitoTracker. Scale bar = 20 μm. (D) Percentage of oocytes with abnormal mitochondrial distribution in the three groups. A total of 74–97 oocytes were analyzed per group. (E) Representative images of spindles and chromosomes in MII oocytes from control, PCOS, and PCOS+NMN groups. Scale bar = 5 μm; scale bar for merged images = 10 μm. (F) Percentage of oocytes with abnormal spindles in the three groups. A total of 55–66 oocytes were counted per group. Graphs represent mean ± SEM from at least three independent experiments. Statistical significance was determined using an unpaired two‐tailed t‐test: **p < 0.01, ***p < 0.001.

3.3. SIRT1 Is a Key Effector of NMN Supplementation in PCOS Oocytes

To investigate the mechanism underlying NMN's effects on PCOS oocytes, we identified 36 downregulated and 14 upregulated DEGs in the PCOS versus control comparison, which were subsequently upregulated and downregulated, respectively, in the PCOS+NMN versus PCOS comparison (Figure 4A,B, Table S3). These overlapping DEGs were defined as “rescue DEGs” and were further validated through quantitative RT‐PCR, conducted independently of the transcriptome analysis (Figure 4C). Enrichment analysis of the rescue DEGs highlighted apoptotic signaling pathways, transcriptional regulation, and mitophagy, among which Sirtuin 1 (Sirt1) emerged as a key factor of interest (Figure 4D,E, Table S4).

FIGURE 4.

Response of Sirt1 to NMN supplementation in PCOS oocytes. (A, B) Venn diagrams showing the overlap of downregulated genes in control vs. PCOS and upregulated genes in PCOS vs. PCOS+NMN (A), as well as the overlap of upregulated genes in control vs. PCOS and downregulated genes in PCOS vs. PCOS+NMN (B). (C) qRT‐PCR results verifying the changes of representative rescue DEGs. 20 oocytes were collected per group. (D, E) Gene Ontology (GO) analysis of biological processes (D) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (E) of rescue DEGs identified in (A) and (B). The graph represents mean ± SEM from at least three independent experiments. Statistical significance was determined using an unpaired two‐tailed t‐test: *p < 0.05, **p < 0.01, ***p < 0.001.

SIRT1 is an NAD⁺‐dependent deacetylase involved in various biological processes related to gamete biology and reproductive medicine [26, 27]. In addition to confirming the undulation of Sirt1 at the transcript level (Figure 4C), an immunoblotting assay demonstrated that the decreased expression of SIRT1 in PCOS MII oocytes was restored by NMN supplementation (Figure 5A,B). A significant reduction in SIRT1 expression was also observed in GV oocytes derived from PCOS mice (Figure S4A,B). To directly assess the functional importance of SIRT1, Sirt1‐flag mRNA and Sirt1 small interfering RNA (siRNA) were respectively microinjected into PCOS GV oocytes. Western blot analysis confirmed exogenous SIRT1 expression (Figure S4C), and quantitative RT‐PCR indicated the down‐regulation of Sirt1 (Figure S4D). It was shown that the effect of SIRT1 expression was similar to that of NMN supplementation in the PCOS oocyte development, manifested as a significant increase in the rates of GVBD and PB1 excretion, which could not be improved with NMN when the expression of Sirt1 was inhibited (Figure 5C–E). Based on the pathways and biological processes associated with Sirt1, we examined apoptotic signaling in PCOS oocytes. The intensity of apoptotic signaling was significantly reduced in PCOS oocytes expressing SIRT1 or adding NMN, but remained unchanged regardless of whether NMN was supplemented or not after Sirt1 knockdown (Figure 5F,G). In summary, SIRT1 is a key mediator in NMN‐induced improvement of PCOS oocyte quality, playing a central role in regulating apoptotic signaling and related pathways.

FIGURE 5.

Role of SIRT1 in the effects of NMN on PCOS oocytes. (A) Western blot analysis of SIRT1 expression in control, PCOS, and PCOS+NMN oocytes at the MII stage. β‐ACTIN was used as a loading control. (B) Quantification of SIRT1 expression among the three groups. A total of 360 oocytes were collected per group. (C) Representative images of PCOS oocytes developing in vitro after SIRT1 overexpression or knockdown. Scale bar = 100 μm. (D, E) Quantitative analysis of GVBD (D) and PB1 extrusion rate (E) in PCOS oocytes with SIRT1 overexpression or knockdown. Total of 80 oocytes were calculated per group. (F) Annexin‐V staining assessing apoptosis in PCOS oocytes with SIRT1 overexpression or knockdown. Scale bar = 20 μm. (G) Quantification of Annexin‐V intensity in PCOS oocytes after SIRT1 overexpression or knockdown. A total of 14–24 oocytes were analyzed per group. All graphs are presented as mean ± SEM by three independent experiments. Statistical significance was determined using an unpaired two‐tailed t‐test: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. NMN Regulates SIRT1 Expression Through the NAD ⁺ Salvage Pathway

The observed fluctuations in SIRT1 expression prompted us to investigate its response to NMN supplementation in PCOS oocytes. We specifically examined the conversion of NMN to NAD⁺, catalyzed by three NMNATs [28]. RNA sequencing analysis showed no significant differences in Nmnat expression among the control, PCOS, and PCOS+NMN groups (Figure S5A). To further explore the role of NMNATs, we knocked down Nmnat expression using siRNA injection (Figure S5B). As a result, NAD⁺ levels remained unchanged in PCOS oocytes, regardless of NMN supplementation (Figure 6A). Correspondingly, Sirt1 expression also failed to be restored by NMN supplementation in Nmnat‐knockdown PCOS oocytes (Figure 6B). Next, we examined the developmental competence of injected GV oocytes matured in vitro. The extrusion rate of the PB1was not improved in the Nmnat‐knockdown group following NMN supplementation, whereas the rate of GVBD was less affected by Nmnat knockdown (Figure 6C–E). Additionally, mitochondrial aggregation abnormalities and spindle defects were not corrected in PCOS oocytes when Nmnat expression was suppressed (Figure S5C–E). These findings suggest that the conversion of NMN to NAD⁺ is essential for Sirt1expression and contributes to the improvement of PCOS oocyte quality through multiple pathways.

FIGURE 6.

Mechanism of NMN supplementation in improving PCOS oocytes. (A) Measurement of NAD⁺ levels in PCOS MII oocytes after knockdown of the three Nmnats. A total of 80 oocytes were collected per group. (B) Detection of Sirt1 expression in PCOS oocytes after Nmnats knockdown. A total of 20 oocytes were analyzed per group. (C) Representative images of PCOS oocytes cultured in vitro after knockdown of the three Nmnats. Scale bar = 100 μm. (D, E) Quantitative analysis of GVBD rate (D) and PB1 extrusion rate (E) in PCOS oocytes after Nmnats knockdown. A total of 80 oocytes were analyzed per group. All graphs represent mean ± SEM from three independent experiments. Statistical significance was determined using an unpaired two‐tailed t‐test: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

In this study, we demonstrated that the poor quality of oocytes from PCOS mice, characterized by elevated ROS, mitochondrial dysfunction, spindle abnormalities, and increased apoptotic signaling, may result from a decline in NAD⁺ levels. The conversion of supplemented NMN into NAD⁺, mediated by NMNAT, effectively alleviates these adverse effects. Among the NAD⁺‐responsive genes, Sirt1 emerged as a critical regulator in PCOS oocytes (Figure 7).

FIGURE 7.

The relationship between NMN supplementation and PCOS oocyte improvement. A schematic illustration shows that exogenous NMN is converted to NAD⁺ through the salvage pathway, leading to increased SIRT1 expression. This, in turn, enhances the quality of PCOS oocytes by reducing oxidative stress, improving mitochondrial function, and stabilizing spindle morphology.

NAD⁺ homeostasis is essential for maintaining proper physiological and biological functions, particularly in mitochondrial activity. As a crucial cofactor, NAD⁺ facilitates hydrogen ion and electron transfer in the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation within mitochondria [29, 30]. A decline in NAD⁺ levels in oocytes from obese and aged mice has been shown to impair mitochondrial function, leading to increased ROS levels and meiotic defects [31, 32, 33]. Our study further confirmed that reduced NAD⁺ levels in PCOS oocytes similarly result in mitochondrial dysfunction, as revealed by transcriptomic analysis and functional validation. Moreover, the enhanced apoptosis and autophagy signaling observed in PCOS oocytes suggests that NAD⁺ depletion severely compromises cellular developmental potential, consistent with findings in granulosa and muscle cells from PCOS models [15, 16].

To restore NAD⁺ homeostasis, the supplementation of NAD⁺ precursors has been extensively studied in various NAD⁺‐deficient conditions [34, 35, 36]. In this study, we demonstrated the pivotal role of NMN in rebalancing NAD⁺ homeostasis in PCOS oocytes, effectively mitigating the detrimental effects caused by NAD⁺ depletion. Notably, we observed an optimal concentration for NMN's regulatory function, which aligns with previous studies on NMN‐mediated cellular protection [23, 37, 38]. One possible explanation is that NMN metabolism is constrained by NMNAT, a rate‐limiting enzyme in the NAD⁺ salvage pathway [39]. Our transcriptomic data revealed stable NMNAT expression across all groups, and Nmnat knockdown significantly reduced NAD⁺ content despite NMN supplementation. These findings suggest that NMN metabolism in oocytes is subject to enzymatic limitations. Additionally, NMN is transported across the plasma membrane via SLC12A8 or incorporated into cells through equilibrative nucleoside transporters after extracellular conversion to nicotinamide riboside [40, 41]. Given that SLC12A8‐mediated NMN transport is sodium‐dependent [40], it is plausible that transporter availability and activity further restrict NMN utilization. However, the precise mechanism of NMN uptake in oocytes remains unclear and warrants further investigation.

In exploring NAD⁺ homeostasis in PCOS oocytes, we identified several genes that exhibited significant fluctuations in response to NAD⁺ changes based on transcriptomic data, and termed rescue DEGs. Among the rescue DEGs, Sirt1 was of particular interest due to its NAD⁺‐dependent regulatory role. Our study confirmed that intracellular NAD⁺ deficiency disrupts the NAD⁺/SIRT1 axis, particularly affecting mitochondrial function and apoptosis [42]. NMN‐mediated SIRT1 activation has been shown to reduce oxidative stress and reverse vascular dysfunction in aged mice [43]. In triple‐negative breast cancer models, NMN‐induced SIRT1 activation led to the deacetylation and functional inactivation of p66Shc, thereby inhibiting tumor growth [44]. Moreover, our findings suggest that the NAD⁺ salvage pathway, regulated by NMNATs, is the primary mechanism by which NMN activates SIRT1.

The application of NMN in human ART has not yet been fully launched, although high doses of NMN are safe for the general population [45, 46]. 10 μmol/L of NMN was used for the development of human early embryos that were 3 pronuclei [47]. However, determining the optimal dosage for ART applications requires further investigation, particularly to ensure no adverse effects on oocyte quality or embryo development. In the future, clinical trials will need to be designed to evaluate the efficacy and safety of NMN in the ART environment.

To sum up, the breakdown of NAD⁺ homeostasis in PCOS oocytes leads to multiple impairments, including mitochondrial dysfunction, oxidative stress, and apoptosis. As a precursor of NAD⁺, NMN restores NAD⁺ balance and enhances oocyte quality and developmental potential through the salvage pathway regulated by NMNAT. Key effector genes, particularly Sirt1, respond dynamically to NAD⁺ fluctuations and play essential regulatory roles. These findings provide valuable insights into potential therapeutic strategies for improving oocyte quality in PCOS patients undergoing assisted reproduction.

Author Contributions

Conceptualization: X.L. and J.Z. Methodology: S.Z., Y.F., X.W., and P.L. Investigation: Y.F., Q.H., X.W., P.L., Y.W., R.Y., C.H., and S.Z. Formal analysis: Y.F., T.G., and S.Z. Validation: C.Z., S.Z., J.Z., and X.L. Visualization: Y.F. and S.Z. Writing: S.Z., C.Z., and Y.F. Funding acquisition: C.Z., S.Z., P.L., and X.L. Resources: C.Z., J.Z., and X.L. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1.

Table S1. RNA Sequencing identified DEGs, related to Figure 2.

Table S2. KEGG and GO of RNA sequencing identified DEGs, related to Figure 2.

Table S3. Rescue genes, related to Figure 4.

Table S4. KEGG and GO of rescue genes, related to Figure 4.

Table S5. Table of primers and siRNA sequences used in this study.

Fan Y., Hu Q., Wu X., et al., “Reverse Effects of Nicotinamide Mononucleotide Supplementation on Declining Quality of Oocytes With Polycystic Ovary Syndrome,” The FASEB Journal 39, no. 14 (2025): e70846, 10.1096/fj.202500921R.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding authors upon reasonable request.