P-255 Nicotinamide mononucleotide (NMN) supplementation during germinal vesicle (GV) rescue could increase the total number of mature oocytes available in women of advanced maternal age (AMA)
Authors: Rodríguez Varela C , Mifsud A , Escrich L , Ramírez N , Buigues A , Pérez S , Albert C , Herraiz S , Pellicer A , Escribá M  J 
Published: July 2024

Abstract

Study question
Does NMN supplementation improve nuclear and cytoplasmic competence of AMA oocytes during GV-rescue?

Summary answer
NMN supplementation enhances GV-rescue efficiency in AMA oocytes by improving nuclear competence and slightly ameliorating cytoplasmic competence.

What is known already
Oocyte competence drastically declines from the age of 35 onwards. With age, organism NAD+ levels also drop, and NAD+ depletion at the oocyte level is associated with lower oocyte and embryo quality. Over the past years, supplementation with the NAD+ precursor nicotinamide mononucleotide (NMN) has been showed to recover oocyte quality and reproductive potential in aged mice due to NAD+ repletion. Thus, we propose the GV-rescue strategy to assess the impact of NMN in vitro supplementation on rescued-oocyte competence, according to maternal age.

Study design, size, duration
Experimental, prospective study performed on GVs collected after controlled ovarian stimulation (COS), oocyte pick-up (OPU) and denudation; from November 2023 to January 2024. After signed informed consent, GVs were randomized into the control and study (NMN) groups, in a ratio 1:1. A total of two or four GVs were collected from each cycle, in order to avoid any cycle-related potential bias.

Participants/materials, setting, methods
Young (≤35y.o) or AMA (&gt;35y.o.) women from an infertility clinic (Spain) donated their GVs. They were cultured in time-lapsed, CSCM-NXC medium with or without 100µM-NMN (37oC,6%CO2,5%O2). Nuclear dynamics (tGVBD, tMI and tMII) were described, using “denudation” as zero-time (t0). Rescued metaphase-II (rMII) were artificially activated (≤2.8h post-rMII; 8uM A23187,5min; 10ug/ml puromycin,5h). Normal oocyte activation (NOA) was assessed: 2ndpolar body (PB) extrusion, one pronucleus. Continuous and categorical variables were compared by paired-samples and χ2 tests, respectively.

Main results and the role of chance
Two hundred and 82 GVs were collected in the young (n = 73 women) and aged groups (n = 35 women), respectively. In the young group, NMN supplementation did not affect the rescue rate (RR=rMII/GV; 54.0%; p = 0.21) but in the AMA-NMN group RR significantly increased (24.4% vs. 48.8%; p = 0.04). Indeed, rMII from both AMA-NMN and young-control groups showed comparable RR (53.1%; p = 0.37) and nuclear dynamics (tGVBD: 5.4±2.8h [95%CI:4.9-5.9], p = 0.10; tMI: 14.8±2.5h [95%CI:14.3-15.2], p = 0.55; t1PB: 20.1±3.2h [95%CI:19.6-20.7], p = 0.33). In contrast, rMII from AMA-control group extruded the 1PB earlier (18.4±2.6h [95%CI: 16.6-20.2) than young-control rMII (p = 0.02). Concerning artificial activation, a slightly non-significant detrimental effect of NMN was observed on NOA rate (rMII with NOA/incubated rMII) in the young group (69.2% vs. 50.0%; p = 0.17). In contrast, NMN-supplement seems to improve the NOA rate in AMA women (33.3% vs. 42.9%; p = 0.78).

Limitations, reasons for caution
Recruitment rate is lower in AMA than in younger women. Hence, the number of GVs included was lower in the AMA group, providing a potential bias in data interpretation. Results from this preliminary analysis should be tested in further studies with a larger sample size.

Wider implications of the findings
GV-rescue strategy might increase the number of MII available. In the particular case of women &gt;35, NMN-GV-rescue protocol improves quantitative and qualitative the rMIIs, which become closer to those from young women. Since GV has not undergone any chromosome segregation, to validate whether NMN-supplementation could assist reliable meiosis are urged.

Trial registration number
2303-VLC-047-ME