Climacteric : the journal of the International Menopause Society · 2026
l-Proline as a safer strategy to preserve ovarian function for patients facing gonadotoxic therapies.
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Du Juan, Ruan Xiangyan, Gu Muqing, Cheng Jiaojiao
Аннотация
OBJECTIVE: To develop a safer strategy for preserving ovarian function in patients facing gonadotoxic therapies, this study evaluated the natural osmolytes l-proline and betaine as dimethyl sulfoxide (DMSO)-free alternatives for human ovarian tissue cryopreservation (OTC).
METHOD: The study first characterized the physicochemical properties of the cryoprotectants (CPAs). Ovarian cortical tissues from 10 patients were then processed into biopsies, which were randomized and cryopreserved using slow freezing with 1.5 M l-proline, betaine or DMSO (control). After thawing and a 4-day in vitro culture, assessment included estradiol secretion, glucose uptake, follicular viability (Calcein-AM), apoptosis (TUNEL), and markers of angiogenesis (CD31) and proliferation (Ki-67).
RESULTS: l-Proline demonstrated cryoprotective efficacy comparable to DMSO in key follicular metrics: estradiol secretion and follicular viability were not significantly different between the l-proline and DMSO groups, and both were superior to betaine. Apoptosis was equally low in the l-proline and DMSO groups, and considerably lower than in the betaine group. In contrast, DMSO showed higher glucose uptake and expression of CD31 and Ki-67.
CONCLUSION: l-Proline is an effective natural alternative to DMSO for OTC, achieving equivalent preservation of the follicular reserve and function with a favorable biosafety profile. These findings highlight its significant translational potential for enhancing the safety profile of clinical fertility preservation.
METHOD: The study first characterized the physicochemical properties of the cryoprotectants (CPAs). Ovarian cortical tissues from 10 patients were then processed into biopsies, which were randomized and cryopreserved using slow freezing with 1.5 M l-proline, betaine or DMSO (control). After thawing and a 4-day in vitro culture, assessment included estradiol secretion, glucose uptake, follicular viability (Calcein-AM), apoptosis (TUNEL), and markers of angiogenesis (CD31) and proliferation (Ki-67).
RESULTS: l-Proline demonstrated cryoprotective efficacy comparable to DMSO in key follicular metrics: estradiol secretion and follicular viability were not significantly different between the l-proline and DMSO groups, and both were superior to betaine. Apoptosis was equally low in the l-proline and DMSO groups, and considerably lower than in the betaine group. In contrast, DMSO showed higher glucose uptake and expression of CD31 and Ki-67.
CONCLUSION: l-Proline is an effective natural alternative to DMSO for OTC, achieving equivalent preservation of the follicular reserve and function with a favorable biosafety profile. These findings highlight its significant translational potential for enhancing the safety profile of clinical fertility preservation.