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Ventayol-Guirado M, Hernandez-Rodriguez J, Florit J, Llull-Alberti MV, Barragan R, Ferragut JF, Martorell J, Heine-Suñer D, Martinez I, Picornell A, Torres-Juan L, Peralta L. First follicular fluid cf-mtDNA sequencing unveils ovarian stimulation-induced mutations impacting oocyte quality and IVF success. J Assist Reprod Genet 2025:10.1007/s10815-025-03511-8. [PMID: 40405035 DOI: 10.1007/s10815-025-03511-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2025] [Accepted: 05/05/2025] [Indexed: 05/24/2025] Open
Abstract
PURPOSE Cell-free mitochondrial DNA (cf-mtDNA) has emerged as a promising non-invasive method to predict embryo implantation potential. This study presents the first sequencing of cf-mtDNA in follicular fluid, aiming to explore the impact of ovarian stimulation treatment on the accumulation of mtDNA mutations and assess their implications for IVF outcomes. METHODS We selected 24 women aged 27 to 35 from the Assisted Reproduction Unit based on inclusion criteria requiring them to be undergoing their first IVF cycle without known infertility issues. For each participant, blood samples were collected prior to ovarian stimulation, and follicular fluid samples were obtained at the time of oocyte retrieval. Long-PCR amplification and next-generation sequencing were used to identify mtDNA mutations. RESULTS Ovarian stimulation-induced mtDNA mutations in follicular fluid were identified primarily in the D-loop region of the mitochondrial genome. These mutations were correlated with low oocyte counts and poor embryo quality, ultimately reducing IVF success rates. The accumulation of these mutations followed a time-dependent pattern, with longer stimulation durations resulting in higher mutation rates and lower pregnancy outcomes. Prolonged stimulations may exacerbate oxidative stress, negatively impacting oocyte quality. CONCLUSIONS By introducing the first sequencing of follicular fluid cf-mtDNA, this study establishes a direct link between ovarian stimulation-induced mtDNA mutations and reduced IVF efficacy, highlighting them as non-invasive biomarkers for predicting IVF outcomes. Future research may focus on optimizing stimulation protocols or incorporating antioxidants to minimize mtDNA mutations, improving oocyte quality and overall IVF success rates for patients.
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Affiliation(s)
- Marc Ventayol-Guirado
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain.
| | - Jessica Hernandez-Rodriguez
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
- Molecular Diagnostics and Clinical Genetics Unit, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
| | - Joana Florit
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
| | - Maria Victoria Llull-Alberti
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
| | - Raquel Barragan
- Assisted Reproduction Unit, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
| | - Joana Francesca Ferragut
- Biology Department, Universitat de Les Illes Balears, 07122, Palma, Balearic Islands, Spain
- Institut Universitari d'Investigació en Ciències de La Salut (IUNICS), 07122, Palma, Balearic Islands, Spain
| | - Jaume Martorell
- Obstetrics and Gynecology Service, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
| | - Damian Heine-Suñer
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
- Molecular Diagnostics and Clinical Genetics Unit, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
| | - Iciar Martinez
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
- Molecular Diagnostics and Clinical Genetics Unit, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
| | - Antonia Picornell
- Biology Department, Universitat de Les Illes Balears, 07122, Palma, Balearic Islands, Spain
- Institut Universitari d'Investigació en Ciències de La Salut (IUNICS), 07122, Palma, Balearic Islands, Spain
| | - Laura Torres-Juan
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
- Molecular Diagnostics and Clinical Genetics Unit, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
| | - Laura Peralta
- Genomics of Health Research Group, Health Research Institute of the Balearic Islands (IdISBa), 07120, Palma, Balearic Islands, Spain
- Assisted Reproduction Unit, Hospital Universitari Son Espases, 07120, Palma, Balearic Islands, Spain
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Pan M, Shi H, Qi T, Cai L, Ge Q. The biological characteristics of long cell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection. Gene 2024; 927:148667. [PMID: 38857715 DOI: 10.1016/j.gene.2024.148667] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 05/30/2024] [Accepted: 06/06/2024] [Indexed: 06/12/2024]
Abstract
An improved understanding of the cfDNA fragmentomics has proved it as a promising biomarker in clinical applications. However, biological characteristics of cfDNA in spent embryos culture medium (SECM) remain unsolved obstacles before the application in non-invasive in-vitro embryo selection. In this study, we developed a Tn5 transposase and ligase integrated dual-library construction sequencing strategy (TDual-Seq) and revealed the fragmentomic profile of cfDNA of all sizes in early embryonic development. The detected ratio of long cfDNA (>500 bp) was improved from 4.23 % by traditional NGS to 12.80 % by TDual-Seq. End motif analysis showed long cfDNA molecules have a more dominance of fragmentation intracellularly in apoptotic cells with higher predominance of G-end, while shorter cfDNA undergo fragmentation process both intracellularly and extracellularly. Moreover, the mutational pattern of cfDNA and the correlated GO biological process were well differentiated in cleavage and blastocyst embryos. Finally, we developed a multiparametric index (TQI) that employs the fragmentomic profiles of cfDNA, and achieved an area under the ROC curve of 0.927 in screening top quality embryos. TDual-Seq strategy has facilitated characterizing the fragmentomic profile of cfDNA of all sizes in SECM, which are served as a class of non-invasive biomarkers in the evaluation of embryo quality in in-vitro fertilization. And this improved strategy has opened up potential clinical utilities of long cfDNA analysis.
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Affiliation(s)
- Min Pan
- School of Medicine, Southeast University, Nanjing, China; State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Huajuan Shi
- State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Ting Qi
- State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Lingbo Cai
- Clinical Center of Reproductive Medicine, State Key Laboratory of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
| | - Qinyu Ge
- State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
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Shafie A, Aleyasin A, Saffari M, Saedi M, Rostami S, Rezayi S, Mohammadi SD, Amidi F. Astaxanthin improves assisted reproductive technology outcomes in poor ovarian responders through alleviating oxidative stress, inflammation, and apoptosis: a randomized clinical trial. J Ovarian Res 2024; 17:212. [PMID: 39482765 PMCID: PMC11526544 DOI: 10.1186/s13048-024-01537-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2024] [Accepted: 10/15/2024] [Indexed: 11/03/2024] Open
Abstract
BACKGROUND Poor ovarian response (POR) to controlled ovarian stimulation (COS) remains challenging, especially in advanced-age women with diminished ovarian reserve, resulting in low live birth rates. Many patients prefer to conceive with their eggs, underscoring the need for improved treatments. This study explores astaxanthin potential as a COS adjuvant to improve ovarian response and assisted reproductive technology (ART) outcomes, considering its impact on oxidative stress (OS), inflammation, and apoptosis, which are key factors in POR. METHODS In this randomized, triple-blind, placebo-controlled trial, 60 infertile POR patients from POSEIDON Group 4 (the poorest prognosis category, age > 35 and poor ovarian reserve (anti-müllerian hormone < 1.2 ng/ml or antral follicle count < 5) undergoing intracytoplasmic sperm injection were enrolled. Patients were assigned to receive either 12 mg/day AST or placebo for eight weeks. All patients underwent a gonadotropin-releasing hormone antagonist regimen for COS. ART outcomes were compared between groups. Blood serum and follicular fluid (FF) were analyzed for OS markers (superoxide dismutase [SOD], total antioxidant capacity [TAC], and malondialdehyde [MDA]), and pro-inflammatory cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and vascular endothelial growth factor [VEGF]) via enzyme-linked immunosorbent assay kits, and cell-free DNA [cfDNA] (apoptotic marker) via ALU quantitative polymerase chain reaction. RESULTS After the intervention, the AST group exhibited a significant elevation in serum (P = 0.013) and TAC (P = 0.030), accompanied by a significant reduction in serum MDA (P = 0.005). No significant differences between AST and placebo groups were observed in OS markers in FF. AST group showed significant reductions in the serum IL-6 (P < 0.001), IL-8 (P = 0.001), and VEGF (P = 0.002) levels following AST therapy. In the AST group, FF levels of IL-6 (P = 0 < 001), IL-8 (P = 0.036), VEGF (P = 0.006), and cfDNA (P < 0.001) were significantly lower than in the placebo group. Between-group comparisons showed significant differences in the alterations of serum SOD (P = 0.027), IL-6 (P < 0.001), and IL-8 (P = 0.035) levels between AST and placebo groups. The AST group showed significant increases in the number of retrieved oocytes (P = 0.003), MII oocytes (P = 0.004), frozen embryos (P = 0.037), and high-quality embryos (P = 0.014) compared to the placebo group. CONCLUSION AST shows promise as a COS adjuvant therapy, potentially enhancing some ART outcomes in POR through alleviating OS, inflammation, and apoptosis. TRIAL REGISTRATION Clinical trial registration number: IRCT20230223057510N1, URL: https://irct.behdasht.gov.ir/trial/68870 , registration date: 2023 March 16.
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Affiliation(s)
- Anahid Shafie
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave, P.O. Box: 1461884513, Tehran, Iran
| | - Ashraf Aleyasin
- Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
- Department of Obstetrics and Gynecology, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Mojtaba Saffari
- Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mojtaba Saedi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave, P.O. Box: 1461884513, Tehran, Iran
| | - Sahar Rostami
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave, P.O. Box: 1461884513, Tehran, Iran
- Department of Obstetrics and Gynecology, Yas Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran
| | - Saeede Rezayi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave, P.O. Box: 1461884513, Tehran, Iran
| | - Seyed Danial Mohammadi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave, P.O. Box: 1461884513, Tehran, Iran
| | - Fardin Amidi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Poorsina Ave, P.O. Box: 1461884513, Tehran, Iran.
- Department of Obstetrics and Gynecology, Yas Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran.
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Kussano NR, Franco MM, Dode MAN. Biochemical profiling of the follicular environment to predict oocyte competence in cattle. PLoS One 2024; 19:e0298316. [PMID: 38466703 PMCID: PMC10927144 DOI: 10.1371/journal.pone.0298316] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 01/18/2024] [Indexed: 03/13/2024] Open
Abstract
To identify markers of oocyte competence, we compared the biochemical characteristics of fluid and cells from follicles containing oocytes with different capacities to form an embryo. Follicles (5-6 mm) were dissected, and follicular fluid (FF), granulosa cells (GC), cumulus cells (CC) from immature and mature cumulus-oocyte-complexes (COC) were individually collected. The oocytes were matured, fertilized, and cultured individually until day 8 (D8) of development. On D8, the samples were grouped according to embryo production into those that gave rise to blastocysts (EMB) and those that did not reach the blastocyst stage (NEMB). In CCs from immature and mature COCs and GCs, expression of CASP3, SERPINE2, VCAN, LUM, FSHR, EGFR, PGR, and GHR genes was quantified. Cell-free DNA (cfDNA), progesterone, and estradiol concentrations in the FF were determined. Data were analyzed by Mann-Whitney U test (GraphPad Prism 9). GHR was highly expressed in immature CCs from the EMB group, whereas CASP3 was highly expressed in mature CCs from the NEMB group (P<0.05). During maturation, the expression of CASP3 and GHR genes increased only in the NEMB group. ART2 cfDNA was highly detected in FF of the NEMB compared to the EMB group. Progesterone concentration was similar between the groups, whereas estradiol concentration was higher (P<0.05) in the EMB than in the NEMB group. It was concluded that a higher level of GHR transcripts in immature CCs, lower CASP3 expression in CCs from matured COCs, lower levels of ART2, and higher estradiol concentrations in FF may indicate oocytes with greater potential for development.
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Affiliation(s)
| | - Mauricio Machaim Franco
- Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Brasília-DF, Brazil
- School of Veterinary Medicine, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil
- Institute of Biotechnology, Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil
| | - Margot Alves Nunes Dode
- Institute of Biology, University of Brasilia, Brasília-DF, Brazil
- Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Brasília-DF, Brazil
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Raad G, Tanios J, Serdarogullari M, Bazzi M, Mourad Y, Azoury J, Yarkiner Z, Liperis G, Fakih F, Fakih C. Mature oocyte dysmorphisms may be associated with progesterone levels, mitochondrial DNA content, and vitality in luteal granulosa cells. J Assist Reprod Genet 2024; 41:795-813. [PMID: 38363455 PMCID: PMC10957819 DOI: 10.1007/s10815-024-03053-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Accepted: 02/01/2024] [Indexed: 02/17/2024] Open
Abstract
PURPOSE To identify whether follicular environment parameters are associated with mature oocyte quality, embryological and clinical outcomes. METHODS This retrospective study examined 303 mature oocytes from 51 infertile women undergoing ICSI cycles between May 2018 and June 2021. Exclusion criteria consisted of advanced maternal age (> 36 years old), premature ovarian failure, obesity in women, or use of frozen gametes. Luteal granulosa cells (LGCs) were analyzed for mitochondrial DNA/genomic (g) DNA ratio and vitality. The relationships between hormone levels in the follicular fluid and oocyte features were assessed. Quantitative morphometric measurements of mature oocytes were assessed, and the association of LGC parameters and oocyte features on live birth rate after single embryo transfer was examined. RESULTS Results indicated an inverse correlation between the mtDNA/gDNA ratio of LGCs and the size of polar body I (PBI). A 4.0% decrease in PBI size was observed with each one-unit increase in the ratio (p = 0.04). Furthermore, a 1% increase in LGC vitality was linked to a 1.3% decrease in fragmented PBI (p = 0.03), and a 1 ng/mL increase in progesterone levels was associated with a 0.1% rise in oocytes with small inclusions (p = 0.015). Associations were drawn among LGC characteristics, perivitelline space (PVS) debris, cytoplasmic inclusions, PBI integrity, and progesterone levels. Certain dysmorphisms in mature oocytes were associated with embryo morphokinetics; however, live birth rates were not associated with follicular parameters and oocyte quality characteristics. CONCLUSION Follicular markers may be associated with mature oocyte quality features.
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Affiliation(s)
- Georges Raad
- Al Hadi Laboratory and Medical Center, Beirut, Lebanon
- Faculty of Medicine and Medical Sciences, Holy Spirit University of Kaslik (USEK), Jounieh, Lebanon
| | | | - Munevver Serdarogullari
- Department of Histology and Embryology, Faculty of Medicine, Cyprus International University, Northern Cyprus Via Mersin 10, Mersin, Turkey
| | - Marwa Bazzi
- Al Hadi Laboratory and Medical Center, Beirut, Lebanon
| | - Youmna Mourad
- Al Hadi Laboratory and Medical Center, Beirut, Lebanon
| | - Joseph Azoury
- Azoury IVF Clinic, ObGyn and Infertility, Beirut, Lebanon
| | - Zalihe Yarkiner
- Faculty of Arts and Sciences-Department of Basic Sciences and Humanities, Cyprus International University, Northern Cyprus Via Mersin 10, Mersin, Turkey
| | - Georgios Liperis
- Westmead Fertility Centre, Institute of Reproductive Medicine, University of Sydney, Westmead, NSW, Australia.
| | - Fadi Fakih
- Al Hadi Laboratory and Medical Center, Beirut, Lebanon
| | - Chadi Fakih
- Al Hadi Laboratory and Medical Center, Beirut, Lebanon
- Faculty of Medicine, Lebanese University, Beirut, Lebanon
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Tsirka G, Zikopoulos A, Papageorgiou K, Kostoulas C, Tsigkas I, Moustakli E, Kaltsas A, Sarafi E, Michaelidis TM, Georgiou I. The Ratio of cf-mtDNA vs. cf-nDNA in the Follicular Fluid of Women Undergoing IVF Is Positively Correlated with Age. Genes (Basel) 2023; 14:1504. [PMID: 37510407 PMCID: PMC10379089 DOI: 10.3390/genes14071504] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Revised: 07/19/2023] [Accepted: 07/21/2023] [Indexed: 07/30/2023] Open
Abstract
Age-related mitochondrial markers may facilitate the prognosis of artificial reproductive technology outcomes. In this report, we present our study concerning the ratio of cf-mtDNA/cf-nDNA, namely the amount of cell-free mitochondrial DNA relative to cell-free nuclear DNA, in the follicular fluid (FF) of women undergoing IVF, aiming to generate a molecular fingerprint of oocyte quality. The values of this ratio were measured and compared among three groups of women (101 in total): (A) 31 women with polycystic ovary syndrome (PCOS), (B) 34 women younger than 36 years, and (C) 36 women older than 35 years of age. Real-time quantitative PCR (qPCR) was performed to quantify the ratio by using nuclear- and mitochondrial-specific primers and analyzed for potential correlation with age and pregnancy rate. Our analysis showed that the level of FF-cf-mtDNA was lower in the group of advanced-age women than in the groups of PCOS and non-PCOS women. Moreover, a significant positive correlation between FF-cf-mtDNA and the number of mature (MII) oocytes was observed. Collectively, the data show that the relative ratio of cf- mtDNA to cf-nDNA content in human FF can be an effective predictor for assessing the corresponding oocyte's age-related performance in IVF.
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Affiliation(s)
- Georgia Tsirka
- Laboratory of Medical Genetics in Clinical Practice, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
| | | | - Kyriaki Papageorgiou
- Department of Biological Applications & Technology, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
- Biomedical Research Institute, Foundation for Research and Technology-Hellas, 45115 Ioannina, Greece
| | - Charilaos Kostoulas
- Laboratory of Medical Genetics in Clinical Practice, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
| | - Ioannis Tsigkas
- Department of Biological Applications & Technology, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
- Biomedical Research Institute, Foundation for Research and Technology-Hellas, 45115 Ioannina, Greece
| | - Efthalia Moustakli
- Laboratory of Medical Genetics in Clinical Practice, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
| | - Aris Kaltsas
- Laboratory of Spermatology, Department of Urology, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
| | - Eleftheria Sarafi
- Department of Biological Applications & Technology, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
- Biomedical Research Institute, Foundation for Research and Technology-Hellas, 45115 Ioannina, Greece
| | - Theologos M Michaelidis
- Department of Biological Applications & Technology, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
- Biomedical Research Institute, Foundation for Research and Technology-Hellas, 45115 Ioannina, Greece
| | - Ioannis Georgiou
- Laboratory of Medical Genetics in Clinical Practice, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece
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Evaluation of the potential of miR-21 as a diagnostic marker for oocyte maturity and embryo quality in women undergoing ICSI. Sci Rep 2023; 13:1440. [PMID: 36697494 PMCID: PMC9876918 DOI: 10.1038/s41598-023-28686-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2022] [Accepted: 01/23/2023] [Indexed: 01/26/2023] Open
Abstract
MicroRNAs are small molecules that play a crucial role in regulating a woman's reproductive system. The present study evaluates the expression of miR-21 in the serum, follicular fluid (FF), and cumulus cells (CCs) and their association with oocyte maturity and embryo quality in women undergoing intracytoplasmic sperm injection. Women subjects were divided into the case (54 Patients with female factor infertility) and control groups (33 patients with male factor infertility). The level of miR-21 was measured using Real-Time PCR. The level of miR-21 was significantly lower in the CCs, FF, and serum in the case compared to the control group (p < 0.05). MiR-21 abundance was higher in FF and CCs samples than in serum. Furthermore, there was a significant increase in CCs to FF in the case group (p < 0.05). A significant decrease in oocyte count, MII oocytes, and percentage of mature oocytes were observed in the case group (p < 0.05). The expression of miR-21 in FF and CCs was positively related to oocyte maturation, but no correlation with embryo development was observed. This study found that miR-21 is expressed less in women with female factor infertility, and human oocytes' development is crucially affected by the expression of miR-21. Therefore, miR-21 could provide new helpful biomarkers of oocyte maturity.
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Cell-Free DNA as a New Biomarker of IVF Success, Independent of Any Infertility Factor, Including Endometriosis. Diagnostics (Basel) 2023; 13:diagnostics13020208. [PMID: 36673018 PMCID: PMC9858053 DOI: 10.3390/diagnostics13020208] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Revised: 12/31/2022] [Accepted: 01/03/2023] [Indexed: 01/09/2023] Open
Abstract
Cell-free DNA fragments detected in blood and in other biological fluids are released from apoptotic/necrotic cells. In this study, we analyzed cfDNA levels in follicular fluid (FF) samples from patients with infertility. Samples were collected from 178 infertile women and cfDNA was extracted and quantified by qPCR, using ALU115 and ALU247 primers, and statistical correlations were performed. We found that cfDNA concentration was significantly higher in FF pools from women aged 35 and over than in women under 35 years of age (p = 0.017). We also found that q247 cfDNA levels were significantly higher in women with an associated female factor, such as endometriosis, PCOS and POF, compared with women with no specific cause of infertility (p = 0.033). The concentration of cfDNA did not vary significantly in each group of women with an associated female factor. The concentration of cfDNA was significantly higher in the FF of women that obtained embryos with a high fragmentation rate, compared to embryos with a low fragmentation rate (p = 0.007). Finally, we found that women who did not become pregnant during IVF treatments had higher q247 cfDNA levels (p = 0.043). The quantification of cfDNA could be an important biomarker of follicular micro-environment quality to predict embryo quality and the success of IVF, making them more specific and effective.
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Jingyun Z, Zhaoyan N, Xianglong K, Liqian, Na Z, Lvcuiting, Lina G, Haifeng W. Study on the relationship between SlRTl and oxidative stress in aged patients undergoing in vitro fertilization and embryo transfer cycles. J Gynecol Obstet Hum Reprod 2023; 52:102516. [PMID: 36442811 DOI: 10.1016/j.jogoh.2022.102516] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2022] [Revised: 11/24/2022] [Accepted: 11/25/2022] [Indexed: 11/27/2022]
Abstract
PURPOSE Oxidative stress (OS) has been suggested to be involved in the pathogenesis of fertility reduction in aged patient. Silent Information Regulator 1 (SIRT1) is involved in antioxidant defense and also plays a role in regulation ovarian function. The purpose of this study was to evaluate SIRT1 and OS markers in follicular fluid (FF) and granulosa cells (GCs) for aged patient undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). METHODS This single-center prospective cohort study was performed from May 2020 to October 2021, including 92 patients undergoing IVF/ICSI at authors' institute. The patients were grouped by age, including group A (< 35 year, n = 28, age range 24-29), group B (35-40 year, n = 33, age range 35-40), and group C (> 40 year, n = 31, age range 41-47). The outcomes included in vitro fertilization and embryo transfer (IVF-ET) results, OS markers and SIRT1 levels. RESULTS Women in group B and group C had a significantly lower estradiol (E2) in the trigger day, retrieved oocytes, mature oocytes, two pronuclei (2PN) and Day3 available embryos than group A. Women in group C had a significantly lower implantation rate and clinical pregnancy rate than group A. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and SIRT1 levels were lower in FF of the group B and group C, whereas the malondialdehyde (MDA) level was higher than that in the group A. Moreover, the MDA levels were correlated negatively with SIRT1 (r = -0.422, P=0.001),while the SOD and GSH-Px was positively correlated with SIRT1 (r = 0.409, P = 0.001 and r = 0.526, P = 0.001). CONCLUSIONS The oxidative stress may be related to the decrease of SIRT1 in aged patients undergoing IVF-ET.
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Affiliation(s)
- Zhao Jingyun
- Department of Reproductive Medicine, the Fourth Hospital of Hebei Medical University, No.12 Jiankang Road, Shijiazhuang, Hebei, China Postal code: 050011
| | - Nie Zhaoyan
- Department of Reproductive Medicine, the Fourth Hospital of Hebei Medical University, No.12 Jiankang Road, Shijiazhuang, Hebei, China Postal code: 050011
| | - Kong Xianglong
- Department of Medical Laboratory, Hebei Chest Hospital, No. 372 Shengli North Street, Shijiazhuang, Hebei, China Postal code: 050010
| | - Liqian
- Department of Medical Laboratory, Hebei Chest Hospital, No. 372 Shengli North Street, Shijiazhuang, Hebei, China Postal code: 050010
| | - Zhang Na
- Department of Reproductive Medicine, the Fourth Hospital of Hebei Medical University, No.12 Jiankang Road, Shijiazhuang, Hebei, China Postal code: 050011
| | - Lvcuiting
- Department of Reproductive Medicine, the Fourth Hospital of Hebei Medical University, No.12 Jiankang Road, Shijiazhuang, Hebei, China Postal code: 050011
| | - Guo Lina
- Department of Reproductive Medicine, the Fourth Hospital of Hebei Medical University, No.12 Jiankang Road, Shijiazhuang, Hebei, China Postal code: 050011
| | - Wu Haifeng
- Department of Medical Laboratory, Hebei Chest Hospital, No. 372 Shengli North Street, Shijiazhuang, Hebei, China Postal code: 050010.
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Yanagihara R, Yamamoto Y, Kawakita T, Noguchi H, Yano Y, Hayashi N, Ohta C, Minato S, Kamada S, Aoki H, Tamura K, Masaki R, Tachibana A, Arakaki R, Yoshida K, Kato T, Irahara M, Iwasa T. Biotin levels in blood and follicular fluid and their associations with pregnancy outcomes in IVF/ICSI patients. THE JOURNAL OF MEDICAL INVESTIGATION 2022; 69:65-69. [PMID: 35466148 DOI: 10.2152/jmi.69.65] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
It has been shown that biotin, a water-soluble vitamin (B7), plays roles in reproductive functions, such as oocyte maturation and embryo development, in experimental animals. On the other hand, little is known about the clinical effects of biotin on human reproduction. In this study, serum and follicular fluid biotin levels were measured in patients who underwent in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), and their associations with reproductive outcomes were evaluated. As a result, biotin was detected in follicular fluid, as well as serum, and the biotin levels of follicular fluid were found to be positively correlated with those of serum. The biotin levels of serum were higher than those of follicular fluid, suggesting that biotin may be taken up into the follicular fluid from the blood. Although serum and follicular fluid biotin levels tended to be higher in pregnant patients than in non-pregnant patients, these data did not show the significant statistical difference. These findings indicate that biotin does not contribute to the maintenance of oocyte quality, and hence, it does not increase fertilization and pregnancy rates. J. Med. Invest. 69 : 65-69, February, 2022.
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Affiliation(s)
- Rie Yanagihara
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Yuri Yamamoto
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Takako Kawakita
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Hiroki Noguchi
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Yuya Yano
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Noriko Hayashi
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Chiaki Ohta
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Saki Minato
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Shuhei Kamada
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Hidenori Aoki
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Kou Tamura
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Rie Masaki
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Ayaka Tachibana
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Ryosuke Arakaki
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Kanako Yoshida
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Takeshi Kato
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Minoru Irahara
- Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
| | - Takeshi Iwasa
- Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
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Vaigauskaitė B, Baušytė R, Valatkaitė E, Skliutė G, Kazėnaitė E, Ramašauskaitė D, Navakauskienė R. Prognostic Gene Predictors of Gestational Diabetes in Endometrium and Follicular Fluid of Women after Infertility. MEDICINA (KAUNAS, LITHUANIA) 2022; 58:498. [PMID: 35454338 PMCID: PMC9025034 DOI: 10.3390/medicina58040498] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 03/22/2022] [Accepted: 03/25/2022] [Indexed: 11/16/2022]
Abstract
Background and objectives. Gestational diabetes mellitus is an increasingly diagnosed metabolic disorder during pregnancy with unknown pathological pathways. Taking into account the growing numbers of women who are conceiving after assisted reproductive technologies, they comprise an engaging target group for gestational diabetes mellitus etiopathogenesis research. In terms of metabolism and genetics, as the evidence shows, both unexplained infertility and gestational diabetes mellitus pose challenges for their interpretation due to the complex bodily processes. Materials and Methods. Our study examined the expression of genes (IGF2, GRB10, CRTC2, HMGA2, ESR1, DLK1, SLC6A15, GPT2, PLAGL1) associated with glucose metabolism in unexplained infertility patients who conceived after in vitro fertilization procedure, were diagnosed with GDM and their findings were compared with control population. Results. There were no significant differences in gene expression of endometrium stromal cells between healthy pregnant women and women with gestational diabetes, although the significant downregulation of CRTC2 was observed in the follicular fluid of women with gestational diabetes mellitus. Moreover, expression of HMGA2 and ESR1 was significantly reduced in FF cells when compared to endometrial cells. Conclusions. These findings may indicate about the importance of follicular fluid as an indicator for gestational diabetes and should be explored more by further research.
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Affiliation(s)
- Brigita Vaigauskaitė
- Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio Av. 7, LT-10257 Vilnius, Lithuania; (R.B.); (E.V.); (G.S.); (R.N.)
- Centre of Obstetrics and Gynaecology of the Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Santariškių St, LT-08661 Vilnius, Lithuania;
| | - Raminta Baušytė
- Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio Av. 7, LT-10257 Vilnius, Lithuania; (R.B.); (E.V.); (G.S.); (R.N.)
- Centre of Obstetrics and Gynaecology of the Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Santariškių St, LT-08661 Vilnius, Lithuania;
| | - Elvina Valatkaitė
- Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio Av. 7, LT-10257 Vilnius, Lithuania; (R.B.); (E.V.); (G.S.); (R.N.)
| | - Giedrė Skliutė
- Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio Av. 7, LT-10257 Vilnius, Lithuania; (R.B.); (E.V.); (G.S.); (R.N.)
| | - Edita Kazėnaitė
- Faculty of Medicine, Vilnius University Hospital Santaros Klinikos, Vilnius University, Santariškių St, LT-08661 Vilnius, Lithuania;
| | - Diana Ramašauskaitė
- Centre of Obstetrics and Gynaecology of the Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Santariškių St, LT-08661 Vilnius, Lithuania;
| | - Rūta Navakauskienė
- Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio Av. 7, LT-10257 Vilnius, Lithuania; (R.B.); (E.V.); (G.S.); (R.N.)
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Nie Z, Zhang N, Guo L, Lv C, Zhang Y, Wang C, Wu H. Growth hormone improved oxidative stress in follicle fluid by influencing Nrf2/Keap1 expression in women of advanced age undergoing IVF. Gynecol Endocrinol 2022; 38:222-226. [PMID: 35112648 DOI: 10.1080/09513590.2021.2003325] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/19/2022] Open
Abstract
OBJECTIVES To investigate whether growth hormone (GH) can improve oxidative stress (OS) by affecting) /nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) in women of advanced age undergoing in vitro fertilization (IVF). METHODS This retrospective study enrolled 141 patients, including 65 aged C patients (patients not treated with GH) and 76 aged GH patients (patients treated with GH). The outcomes included IVF-ET results, OS markers in follicle fluid (FF) and Nrf2 and Keap1 mRNA and protein expressions in granulosa cells (GCs). RESULTS The results showed that GH improved the available blastocyst (p=.047) and implantation rate (p=.043) in women of advanced age undergoing IVF. The malondialdehyde (MDA) content of FF was significantly higher in the aged-C group than in the aged-GH group (p=.013). The antioxidant enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) were significantly lower in the aged-C group than in the aged-GH group (p= .000, p= .049, p= .012 respectively). Nrf2 mRNA and protein expression was significantly higher and Keap1 mRNA and protein expression was lower in the aged-GH group than in the aged-C group (p= .000, p= .000 respectively). CONCLUSIONS The study showed that GH improved embryo quality and implantation rate and alleviated OS in FF, which may be related to Nrf2/Keap1.
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Affiliation(s)
- Zhaoyan Nie
- Department of reproductive medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
| | - Na Zhang
- Department of reproductive medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
| | - Lina Guo
- Department of reproductive medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
| | - Cuiting Lv
- Department of reproductive medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
| | - Yi Zhang
- Department of reproductive medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
| | - Congmin Wang
- Department of reproductive medicine, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
| | - Haifeng Wu
- Department of medical laboratory, Hebei Chest Hospital, Shijiazhuang, China
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14
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Azzam M, Hamood A, Abdulkadim H. Cell-free DNA in Human Follicular Fluid as Biomarker for Intracytoplasmic Sperm Injection Procedure Outcome. Open Access Maced J Med Sci 2021. [DOI: 10.3889/oamjms.2021.7810] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
Background: Follicular fluid considered as an important microenvironment for oocyte development, cell free-DNA (cfDNA) fragments that are found in this fluid and are released from cell apoptosis and/or necrosis, aimed to quantified the level of cf-DNA, in the follicular fluid and to assess any relation between the level of cf-DNA in this fluid with women’s age, duration of infertility, cause of infertility, her ovarian reserve values. Methods: Eighty-nine women were prospectively included in this study FF cf-DNA which was determined by conventional real time PCR-syber green detection approach which quantified by ALU-specific primers. Results: cell-free DNA (cfDNA) level in Follicular fluid samples of Iraqi women level was; cfDNA (Mean±SD, 0.916±0.106 ng/μl). there was no significant relation between cfDNA and pregnancy outcome, but very low level and very high level cf DNA were related to negative pregnancy outcome, cfDNA was second most important predictive factor of pregnancy outcome after fertilization rate, but both not statistically significant p value was (0.622 and 0.241) respectively. Conclusion: current study notice that cfDNA in the follicular fluid may mainly reflect the cellular activity and the balance between programed apoptosis and cell necrosis.
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Pan M, Shi H, Liu Z, Dong J, Cai L, Ge Q. The integrity of cfDNA in follicular fluid and spent medium from embryo culture is associated with embryo grade in patients undergoing in vitro fertilization. J Assist Reprod Genet 2021; 38:3113-3124. [PMID: 34820723 DOI: 10.1007/s10815-021-02357-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 11/12/2021] [Indexed: 11/28/2022] Open
Abstract
PURPOSE This study was conducted to verify if the cfDNA integrity (cfDI) in follicular fluid and subsequent spent embryo medium (SEM) could serve as potential non-invasive biomarker for high-grade embryo selection during IVF/ICSI. METHODS Thirty-two follicular fluids, 32 subsequent corresponding cleavage embryo SEM, and 23 subsequent blastocyst SEM were collected from 11 patients undergoing IVF/ICSI. CfDI was measured by ALU gene amplicons with different sizes by qPCR, as the ratio of long to short fragments. RESULTS CfDI in follicular fluid corresponding to subsequent high-grade cleavage embryos and blastocysts was significantly lower than that related to low-grade embryos (p = 0.018). Conversely, cfDI in SEM was significantly and positively correlated with high-grade embryos at both stages (p = 0.009). ROC curves of the analysis of cfDI in follicular fluid showed great potential in predicting subsequent embryogenesis and embryo grade (AUC > 0.927). Regardless of the cleavage embryo grade by morphology, cfDI in day 3 SEM could predict if the cleavage embryo could develop to a high-grade blastocyst (AUC = 0.820). A concordant shift pattern of cfDI from follicular fluid to subsequent day 3 SEM and day 5 SEM was found in 81.82% participants featured by various clinical characteristics. CONCLUSION CfDI in follicular fluid and SEM was significantly correlated with embryogenesis and embryo grade and could serve as a potential non-invasive biomarker in high-grade embryo selection. Direct qPCR was proved as a labor-saving and sensitive method for the analysis of cfDI in low volume of SEM.
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Affiliation(s)
- Min Pan
- School of Medicine, Southeast University, Nanjing, China
| | - Huajuan Shi
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Zhiyu Liu
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Juan Dong
- Clinical Center of Reproductive Medicine, State Key Laboratory of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - Lingbo Cai
- Clinical Center of Reproductive Medicine, State Key Laboratory of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
| | - Qinyu Ge
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
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16
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Raad G, Tanios J, Kerbaj S, Mourad Y, Fakih F, Shamas F, Azouri J, Fakih C. Stress Management during the Intracytoplasmic Sperm Injection Cycle May Slow Down First Embryo Cleavage and Accelerate Embryo Compaction: A Pilot Randomized Controlled Trial. PSYCHOTHERAPY AND PSYCHOSOMATICS 2021; 90:119-126. [PMID: 33333526 DOI: 10.1159/000512530] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/29/2020] [Accepted: 10/21/2020] [Indexed: 11/19/2022]
Abstract
INTRODUCTION A firm consensus on the effectiveness of psychological interventions during infertility treatment has not been reached yet in terms of mental health and pregnancy rates. Moreover, the influence of these interventions on embryo cleavage kinetics has not been investigated. OBJECTIVE The aim of this work was to study whether stress management in couples undergoing an intracytoplasmic sperm injection (ICSI) cycle influences stress levels, mitochondrial DNA (mtDNA) levels in granulosa cells, and cleavage-stage embryos. METHODS Infertile couples were randomized into a treatment as usual (TAU) group (n = 30) and stress management program (SMP) group (n = 29) at the beginning of an ICSI cycle. Couples in the SMP group attended education and relaxation sessions at each visit to the clinic for folliculometry. The perceived stress scale (PSS) was used to assess stress levels at the beginning and end of the cycle. Moreover, mtDNA levels of granulosa cells and embryo morphokinetics were evaluated. RESULTS Post-intervention, women in the SMP group had significantly lower PSS scores than their initial PSS (p < 0.001; effect size, ES = 0.5) and than the final PSS of the TAU group (p = 0.02; ES = 0.09). Additionally, mtDNA levels were significantly lower in luteal granulosa cells of the SMP group than the TAU group (p = 0.02). An earlier time of pronuclei appearance (p = 0.03) and time to 2 cells (p = 0.015) and a faster time to full compaction (p = 0.045) were detected in the embryos of the SMP group compared with the TAU group. CONCLUSION(S) The implemented program may reduce stress levels, retard first embryo cleavage, and accelerate embryo compaction. Further studies with an active control group are needed to confirm these results.
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Affiliation(s)
- Georges Raad
- Al-Hadi Laboratory and Medical Center, Beirut, Lebanon,
| | | | - Simone Kerbaj
- Azoury IVF Clinic, Mount-Lebanon Hospital, Hazmieh, Lebanon
| | - Youmna Mourad
- Al-Hadi Laboratory and Medical Center, Beirut, Lebanon
| | - Fadi Fakih
- Al-Hadi Laboratory and Medical Center, Beirut, Lebanon
| | - Fatmeh Shamas
- Al-Hadi Laboratory and Medical Center, Beirut, Lebanon
| | - Joseph Azouri
- Azoury IVF Clinic, Mount-Lebanon Hospital, Hazmieh, Lebanon
| | - Chadi Fakih
- Al-Hadi Laboratory and Medical Center, Beirut, Lebanon.,Obstetrics and Gynecology Department, Faculty of Medicine, Lebanese University, Beirut, Lebanon
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17
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Schobers G, Koeck R, Pellaers D, Stevens SJC, Macville MVE, Paulussen ADC, Coonen E, van den Wijngaard A, de Die-Smulders C, de Wert G, Brunner HG, Zamani Esteki M. Liquid biopsy: state of reproductive medicine and beyond. Hum Reprod 2021; 36:2824-2839. [PMID: 34562078 PMCID: PMC8523207 DOI: 10.1093/humrep/deab206] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 08/06/2021] [Indexed: 01/23/2023] Open
Abstract
Liquid biopsy is the process of sampling and analyzing body fluids, which enables non-invasive monitoring of complex biological systems in vivo. Liquid biopsy has myriad applications in health and disease as a wide variety of components, ranging from circulating cells to cell-free nucleic acid molecules, can be analyzed. Here, we review different components of liquid biopsy, survey state-of-the-art, non-invasive methods for detecting those components, demonstrate their clinical applications and discuss ethical considerations. Furthermore, we emphasize the importance of artificial intelligence in analyzing liquid biopsy data with the aim of developing ethically-responsible non-invasive technologies that can enhance individualized healthcare. While previous reviews have mainly focused on cancer, this review primarily highlights applications of liquid biopsy in reproductive medicine.
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Affiliation(s)
- Gaby Schobers
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Rebekka Koeck
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Dominique Pellaers
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Servi J C Stevens
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands
| | - Merryn V E Macville
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands
| | - Aimée D C Paulussen
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Edith Coonen
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Center for Reproductive Medicine, Maastricht University Medical Centre+, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Arthur van den Wijngaard
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands
| | - Christine de Die-Smulders
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Guido de Wert
- Faculty of Health, Medicine and Life Sciences, Department of Health, Ethics and Society, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Han G Brunner
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.,Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
| | - Masoud Zamani Esteki
- Department of Clinical Genetics, Maastricht University Medical Centre+, Maastricht, The Netherlands.,Department of Genetics and Cell Biology, GROW School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands
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Bódis J, Sulyok E, Várnagy Á, Prémusz V, Gödöny K, Makai A, Szenczi Á, Varjas T. Expression of mRNAs for pro-and anti-apoptotic factors in granulosa cells and follicular fluid of women undergoing in vitro fertilization. A pilot study. BMC Pregnancy Childbirth 2021; 21:399. [PMID: 34030662 PMCID: PMC8142473 DOI: 10.1186/s12884-021-03834-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2021] [Accepted: 04/26/2021] [Indexed: 11/10/2022] Open
Abstract
Background This observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF). Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3 ± 5.8 years, body mass index: 24.02 ± 3.12 kg/m2, duration of infertility: 4.2 ± 2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r = 0.393, p = 0.029), but the day of embryo transfer was negatively associated with GC LHR (r = − 0.414, p = 0.020) and GC FSHR transcripts (r = − 0.535, p = 0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs. Conclusion Our study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.
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Affiliation(s)
- József Bódis
- ELKH-PTE Human Reproduction Scientific Research Group, University of Pécs, Édesanyák u. 17., Pécs, H-7624, Hungary
| | - Endre Sulyok
- Doctoral School of Health Sciences, Faculty of Health Sciences, University of Pécs, Vörösmarty u. 4., Pécs, H-7621, Hungary.
| | - Ákos Várnagy
- ELKH-PTE Human Reproduction Scientific Research Group, University of Pécs, Édesanyák u. 17., Pécs, H-7624, Hungary.,Department of Obstetrics and Gynaecology, Medical School, University of Pécs, Édesanyák u. 17., Pécs, H-7624, Hungary
| | - Viktória Prémusz
- ELKH-PTE Human Reproduction Scientific Research Group, University of Pécs, Édesanyák u. 17., Pécs, H-7624, Hungary.,Doctoral School of Health Sciences, Faculty of Health Sciences, University of Pécs, Vörösmarty u. 4., Pécs, H-7621, Hungary
| | - Krisztina Gödöny
- Department of Obstetrics and Gynaecology, Medical School, University of Pécs, Édesanyák u. 17., Pécs, H-7624, Hungary
| | - Alexandra Makai
- Doctoral School of Health Sciences, Faculty of Health Sciences, University of Pécs, Vörösmarty u. 4., Pécs, H-7621, Hungary
| | - Ágnes Szenczi
- Department of Public Health Medicine, Medical School, University of Pécs, Szigeti u. 12., Pécs, H-7621, Hungary
| | - Timea Varjas
- Department of Public Health Medicine, Medical School, University of Pécs, Szigeti u. 12., Pécs, H-7621, Hungary
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Cell-free mtDNA level and its biomarker potency for ART outcome are different in follicular fluid of PCOS and non-PCOS women. Mitochondrion 2021; 59:30-36. [PMID: 33839320 DOI: 10.1016/j.mito.2021.04.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2020] [Revised: 04/03/2021] [Accepted: 04/05/2021] [Indexed: 11/22/2022]
Abstract
INTRODUCTION Lack of reliable biomarkers for estimating the outcome is one of the current gaps in ART. In this study, we assessed whether cell-free mitochondrial DNA within the follicular fluid (FF cf-mtDNA) of PCOS patients has biomarker applicability or not. Furthermore, probable involved mechanisms in the FF cf-mtDNA pathway were evaluated. METHODS The level of FF cf-mtDNA was compared between 50 PCOS patients and 50 women without any certain reproductive disorder, and analyzed for correlations with ART outcome. The associations between levels of FF cf-mtDNA and TFAM, POLG, and RNase H1 genes expression in mural granulosa cells (MGCs), as well as IL-6, and TNFα in follicular fluid (FF) were assessed. RESULTS We identified that FF cf-mtDNA level was significantly lower in PCOS women and was accompanied by a reduction in the expression of mtDNA biogenesis genes in MGCs of the patients. Although a significant association between FF cf-mtDNA level and ART outcome was observed in the control group, no correlation could be proved in the PCOS group. Moreover, the expression level of TFAM was negatively associated, while amounts of IL-6 and TNFα were positively correlated with FF cf-mtDNA level in both groups. CONCLUSION PCOS patients present a lower FF cf-mtDNA level in comparison with non-PCOS women. FF cf-mtDNA has biomarker applicability for ART outcome in women without any certain reproductive disorder, but not for those with PCOS. It seems that mtDNA packaging dysfunction results in elevated FF cf-mtDNA, and subsequent effects are triggered by increasing the inflammatory cytokines.
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Qasemi M, Mahdian R, Amidi F. Cell-free DNA discoveries in human reproductive medicine: providing a new tool for biomarker and genetic assays in ART. J Assist Reprod Genet 2021; 38:277-288. [PMID: 33421023 PMCID: PMC7884523 DOI: 10.1007/s10815-020-02038-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 12/13/2020] [Indexed: 02/02/2023] Open
Abstract
Cell-free DNAs (cfDNAs) are fragmented forms of DNA that are released into extracellular environments. Analyzing them, regarding either concentration or genetic/epigenetic status can provide helpful information about disorders, response to treatments, estimation of success rates, etc. Moreover, since they are presented in body fluids, evaluation of the aforementioned items would be achieved by less/non-invasive methods. In human reproduction field, it is required to have biomarkers for prediction of assisted reproduction techniques (ART) outcome, as well as some non-invasive procedures for genetic/epigenetic assessments. cfDNA is an appropriate candidate for providing the both approaches in ART. Recently, scientists attempted to investigate its application in distinct fields of reproductive medicine that resulted in discovering its applicability for biomarker and genetic/epigenetic analyses. However, due to some limitations, it has not reached to clinical administration yet. In this article, we have reviewed the current reported data with respect to advantages and limitations of cfDNA utilization in three fields of ART, reproduction of male and female, as well as in vitro developed embryos.
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Affiliation(s)
- Maryam Qasemi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Reza Mahdian
- Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran
| | - Fardin Amidi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
- Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
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21
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Huo P, Zhang N, Zhang P, Wu X. The levels of follicular fluid cell-free mitochondrial DNA could serve as a biomarker for pregnancy success in patients with small ovarian endometriosis cysts: A case-control study. Medicine (Baltimore) 2020; 99:e23348. [PMID: 33235102 PMCID: PMC7710174 DOI: 10.1097/md.0000000000023348] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2020] [Revised: 10/19/2020] [Accepted: 10/26/2020] [Indexed: 11/26/2022] Open
Abstract
Ovarian endometriosis cyst (OEC) is caused by the growth of ectopic endometrium into the ovarian cortex, leading to disrupted ovarian cortical structures and infertility. Large OECs are usually surgically removed, and assisted reproductive technology (ART) is required for future pregnancy. The oocyte reserve and development of patients with small non-surgical OECs are unknown. In this study, we compared mitochondrial abnormality, ATPase and IF1 mRNA expression levels, and OXPHO complex proteins between OEC vs control mural granulosa cells (mGCs).OEC mGCs show fewer mitochondria per cell, a higher proportion of aberrant morphology, lower ATPase mRNA levels, higher IF1 mRNA levels, and impaired expression of 3 of the 5 critical proteins involved in the OXPHOS complex, compared with control mGCs. Cell-free mitochondrial DNA (cfmtDNA) levels are higher in the follicular fluid of patients with OEC and were inversely associated with the expression of mtDNA in mGCs and cumulus granulosa cells (cGCs).Taken together, this study indicates that small non-surgical OECs lead to poor quality of oocytes and subsequent embryos during ART compared with control, which was accompanied by mGC mitochondrial dysfunction. mGC and cGC mtDNA and FF cfmtDNA might serve as efficient biomarkers for the non-invasive prediction of pregnancy outcomes in patients with OEC undergoing ART.
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Affiliation(s)
- Ping Huo
- Hebei Medical University, Shijiazhuang
- Department of Reproductive Genetics, Hebei General Hospital, Shijiazhuang
| | - Ning Zhang
- Department of Reproductive Genetics, Hebei General Hospital, Shijiazhuang
| | - Pingping Zhang
- Department of Reproductive Genetics, Hebei General Hospital, Shijiazhuang
| | - Xiaohua Wu
- Hebei Medical University, Shijiazhuang
- Reproductive Medicine Center, Shijiazhuang Obstetrics and Gynecology Hospital, Shijiazhuang, Hebei, China
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22
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Sfakianoudis K, Tsioulou P, Maziotis E, Grigoriadis S, Glava A, Nitsos N, Giannelou P, Makrakis E, Pantou A, Rapani A, Koutsilieris M, Mastorakos G, Pantos K, Simopoulou M. Investigating apoptotic, inflammatory, and growth markers in poor responders undergoing natural in vitro fertilization cycles: a pilot study. Ann N Y Acad Sci 2020; 1489:78-90. [PMID: 33188643 DOI: 10.1111/nyas.14517] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 08/12/2020] [Accepted: 10/07/2020] [Indexed: 11/28/2022]
Abstract
This study investigates follicular fluid (FF) from patients with poor and normal ovarian response undergoing natural assisted reproductive technology cycles. We report about (1) cell-free DNA (cfDNA), which reflects apoptosis; (2) corticotropin-releasing hormone (CRH); (3) interleukin (IL)-15, which reflects inflammation; (4) granulocyte colony-stimulating factor (G-CSF); (5) vascular endothelial growth factor (VEGF); and (6) insulin-like growth factor I (IGF-I), which reflects follicular growth. Forty-four poor responders and 44 normal responders-according to the Bologna criteria-were recruited. FF samples were prepared for cfDNA quantification employing Q-PCR and for CRH, IL-15, G-CSF, VEGF, and IGF-I quantification employing ELISA. Statistically nonsignificant different levels of FF cfDNA, CRH, IL-15, VEGF, and IGF-I were observed. Interestingly, statistically significant higher G-CSF levels were observed in normal responders (302.48 ± 474.36 versus 200.10 ± 426.79 pg/mL, P = 0.003). Lower cfDNA integrity was observed in cycles resulting in clinical pregnancy for both groups (normal: 0.07 ± 0.04 versus 0.25 ± 0.17 ng/μL, P < 0.001; poor: 0.10 ± 0.06 versus 0.26 ± 0.12 ng/μL, P < 0.001). The results predominantly showcase similarities between normal and poor responders pertaining to inflammatory, apoptotic, and growth factors. This may be attributed to the employment of natural cycles in order to exclude controlled ovarian stimulation as a factor-indicating its detrimental effect. As G-CSF levels presented significantly higher in normal responders, its vital role in understanding a compromised ovarian response is highlighted.
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Affiliation(s)
| | - Petroula Tsioulou
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.,Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Evangelos Maziotis
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.,Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Sokratis Grigoriadis
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.,Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Argyro Glava
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Nikolaos Nitsos
- Genesis Athens Clinic, Center for Human Reproduction, Athens, Greece
| | - Polina Giannelou
- Genesis Athens Clinic, Center for Human Reproduction, Athens, Greece.,Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Evangelos Makrakis
- Third Department of Obstetrics and Gynecology, Attikon Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Agni Pantou
- Genesis Athens Clinic, Center for Human Reproduction, Athens, Greece
| | - Anna Rapani
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.,Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Michael Koutsilieris
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - George Mastorakos
- Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | | | - Mara Simopoulou
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.,Second Department of Obstetrics and Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
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23
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Di Pizio P, Celton N, Menoud PA, Belloc S, Cohen Bacrie M, Belhadri-Mansouri N, Rives N, Cabry R, Benkhalifa M. Seminal cell-free DNA and sperm characteristic's: An added biomarker for male infertility investigation. Andrologia 2020; 53:e13822. [PMID: 33040391 DOI: 10.1111/and.13822] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 07/08/2020] [Accepted: 07/26/2020] [Indexed: 12/24/2022] Open
Abstract
Cell-free DNA (Cf-DNA) fragments may constitute an easy-to-measure molecular tool for guiding the choice of care provided to infertile couples who benefit assisted reproductive technology (ART) programmes. Data on Cf-DNA levels in the seminal plasma of men with sperm alterations are scarce. The objective of the present study was to quantify the presence of Cf-DNA in semen by using a quantitative real-time PCR. We compared men with abnormal sperm characteristics (n = 21) with normospermic controls (n = 21). The PCR assay evidenced significantly higher mean Cf-DNA levels in patients with sperm abnormalities than in controls (2.09 versus 1.18 µg/ml, respectively; p = .0003). The Cf-DNA levels were notably higher in men with azoospermia (3.65 µg/ml, versus 1.34 µg/ml in matched controls; p = .03) and men with teratozoospermia (1.80 µg/ml, versus 1.29 µg/ml in matched controls; p = .008). Our data report a significant association between elevated Cf-DNA levels and sperm abnormalities. These results may open up new diagnostic and prognostic perspectives in male infertility.
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Affiliation(s)
- Pierre Di Pizio
- Reproductive Medicine & Biology, Amiens University, Amiens, France.,Reproductive Biology Laboratory, University Hospital Centre Rouen, Rouen, France
| | - Noemie Celton
- Reproductive Medicine & Biology, Amiens University, Amiens, France
| | - Pierre Alain Menoud
- Molecular Diagnostic Laboratory, Unilabs Laboratoire d'Analyses Medicales SA, Genève, Switzerland
| | - Stéphanie Belloc
- Laboratoire CERBA, Laboratoire Lavergne, Saint Ouen l'Aumone, France
| | | | | | - Nathalie Rives
- Reproductive Biology Laboratory, University Hospital Centre Rouen, Rouen, France
| | - Rosalie Cabry
- Reproductive Medicine & Biology, Amiens University, Amiens, France
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24
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Gong Y, Zhang K, Xiong D, Wei J, Tan H, Qin S. Growth hormone alleviates oxidative stress and improves the IVF outcomes of poor ovarian responders: a randomized controlled trial. Reprod Biol Endocrinol 2020; 18:91. [PMID: 32891163 PMCID: PMC7487463 DOI: 10.1186/s12958-020-00648-2] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Accepted: 08/27/2020] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND Oxidative stress (OS), defined as an imbalance between excessive reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) production and antioxidant insufficiency, has been suggested to be involved in the pathogenesis of poor ovarian response (POR). Growth hormone (GH) can reduce OS in some cell types. This study investigated whether GH can improve OS and the in vitro fertilization and embryo transfer (IVF-ET) outcomes of poor ovarian responders. METHODS This study enrolled 105 patients with POR and 58 patients without POR (controls) who were diagnosed according to the Bologna criteria and underwent conventional IVF-ET. Poor ovarian responders were randomly assigned to two groups: the POR-GH group, which received pretreatment with GH 4 IU/d on day 2 of the previous menstrual cycle before IVF until the trigger day, and the POR-C group, which received no pretreatment. OS markers in follicular fluid (FF), ROS levels in granulosa cells (GCs), and the IVF outcomes of the groups were compared. RESULTS Endometrial thickness on trigger day, the number of cleaved embryos, the number of higher-quality embryos, and the rates of embryo formation, higher-quality embryo formation, implantation and clinical pregnancy were significantly increased in the POR-GH group compared with the POR-C group (P < 0.05). Moreover, compared to those in the non-POR group, FF malondialdehyde (MDA), total oxidant status (TOS), oxidative stress index (OSI) and ROS levels in GCs were significantly higher, whereas superoxide dismutase (SOD) and the total antioxidant capacity (TAC) were significantly lower in the POR-C group (P < 0.05). Furthermore, compared with those in the POR-C group, the FF TAC was significantly increased in the POR-GH group, and TOS, OSI and intracellular ROS levels were significantly reduced (P < 0.05). CONCLUSIONS Pretreatment with GH alleviates OS and improves oocyte quality and IVF outcomes of poor ovarian responders. TRIAL REGISTRATION Chinese Clinical Trial Registry. ChiCTR1900021269 . Registered 8 February 2019, http://www.chictr.org.cn/edit.aspx?pid=35837&htm=4 .
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Affiliation(s)
- Yan Gong
- Reproductive Medicine Centre, Sichuan Provincial Women's and Children's Hospital, The Affiliated Women's and children's Hospital of Chengdu Medical College, #290 Shayan West Second Street, Wuhou District, Chengdu, Sichuan, People's Republic of China.
| | - Kun Zhang
- Department of Genetics, School of Bioscience and Technology, Chengdu Medical College, #783 Xindu Avenue, Xindu District, Chengdu, Sichuan, 610500, People's Republic of China.
| | - Dongsheng Xiong
- Reproductive Medicine Centre, Sichuan Provincial Women's and Children's Hospital, The Affiliated Women's and children's Hospital of Chengdu Medical College, #290 Shayan West Second Street, Wuhou District, Chengdu, Sichuan, People's Republic of China
| | - Jiajing Wei
- Reproductive Medicine Centre, Sichuan Provincial Women's and Children's Hospital, The Affiliated Women's and children's Hospital of Chengdu Medical College, #290 Shayan West Second Street, Wuhou District, Chengdu, Sichuan, People's Republic of China
| | - Hao Tan
- Department of Genetics, School of Bioscience and Technology, Chengdu Medical College, #783 Xindu Avenue, Xindu District, Chengdu, Sichuan, 610500, People's Republic of China
| | - Shengfang Qin
- Department of Medical Genetics and Prenatal Diagnosis, Sichuan Provincial Women's and Children's Hospital, The Affiliated Women's and children's Hospital of Chengdu Medical College, Chengdu, Sichuan, People's Republic of China
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25
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Konstantinos S, Petroula T, Evangelos M, Polina G, Argyro G, Sokratis G, Anna R, Andrianos N, Agni P, Michael K, Konstantinos P, George M, Mara S. Assessing the practice of LuPOR for poor responders: a prospective study evaluating follicular fluid cfDNA levels during natural IVF cycles. J Assist Reprod Genet 2020; 37:1183-1194. [PMID: 32221790 DOI: 10.1007/s10815-020-01743-4] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2019] [Accepted: 03/12/2020] [Indexed: 01/08/2023] Open
Abstract
PURPOSE The aim of this study is to provide data on the practice of Luteal Phase Oocyte Retrieval (LuPOR). The authors assess cell-free DNA levels in follicular fluid (ff cfDNA) from poor responders undergoing natural cycles, and comparing it to respective data originating from follicular phase oocyte retrievals. METHODS Forty-seven women were eligible for this prospective study. Participants were classified as poor responders based on Bologna criteria while being detected with a second follicular wave. Follicular fluid was collected and prepared for cfDNA extraction. Levels of cfDNA were quantified via Q-PCR employing the ALU115 and ALU247 primers. These primers are associated with apoptotic and necrotic events. Levels of ff cfDNA resulting from follicular phase oocyte retrieval (FoPOR) and LuPOR-performed in a single menstrual cycle were associated with the number and maturation status of yielded oocytes and the number and fertilization status of resulting zygotes. Survival rate following thawing of cryopreserved zygotes, along with the resulting number of cleavage stage and blastocyst stage embryos are provided. RESULTS Mean levels of ALU115 were significantly lower during FoPOR when compared to LuPOR (0.79 ± 0.72 vs 1.46 ± 1.59 ng/μl, p = 0.02). Regarding the FoPOR group, a significant positive correlation of serum estradiol and ALU115 concentration (p = 0.04) was revealed. A significant negative correlation between serum estradiol and cfDNA integrity was observed both during FoPOR (p = 0.03) and LuPOR (p = 0.03). A significant lower number of retrieved (1.09 ± 0.28 vs 1.29 ± 0.58, p = 0.02) and MII oocytes (0.77 ± 0.55 vs 1.08 ± 0.61, p = 0.02) was observed when comparing the FoPOR to LuPOR groups respectively. The integrity of cfDNA was observed to be higher in FoPOR originating embryos that arrested either prior to cleavage (0.28 ± 0.13 vs 0.17 ± 0.10, p = 0.006) or prior to blastocyst formation (0.28 ± 0.12 vs 0.13 ± 0.06, p = 0.04). In the case of LuPOR originating embryos, cfDNA integrity was observed to be higher in embryos that arrested only prior to the blastocyst stage (0.27 ± 0.20 vs 0.11 ± 0.07, p = 0.008). Similarly, cfDNA integrity was observed to be lower in top quality blastocysts originating from FoPOR (0.07 ± 0.04 vs 0.17 ± 0.05, p = 0.03) and in top quality cleavage stage embryos (0.09 ± 0.06 vs 0.31 ± 0.22, p = 0.01) and blastocysts (0.06 ± 0.02 vs 0.14 ± 0.06, p = 0.02) originating from LuPOR. CONCLUSIONS Our results indicate that ff originating from LuPOR presents with higher levels of cfDNA. The higher cfDNA levels are attributed to mainly apoptotic events, as the ALU247 levels and DNA integrity did not differ statistically significantly between FoPOR and LuPOR. The absolute mean level of ALU247 corresponding to necrotic events was higher in LuPOR. Regarding embryological data, cfDNA integrity was correlated with both number and quality of cleavage stage embryos in both FoPOR and LuPOR, along with blastocyst stage embryos in LuPOR. Necrotic events were associated with poorer blastocyst formation rate and blastocyst quality in LuPOR. As the comparison between FoPOR and LuPOR results to similar IVF laboratory data, the practice of LuPOR may stand as a promising approach for poor responders, while it merits further investigation.
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Affiliation(s)
| | - Tsioulou Petroula
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Maziotis Evangelos
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Giannelou Polina
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Glava Argyro
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Grigoriadis Sokratis
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Rapani Anna
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Nezos Andrianos
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Pantou Agni
- Genesis Athens Clinic, Centre for Human Reproduction, Athens, Greece
| | - Koutsilieris Michael
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | | | - Mastorakos George
- Assisted Conception Unit, 2nd Department of Obstetrics & Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Simopoulou Mara
- Department of Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
- Assisted Conception Unit, 2nd Department of Obstetrics & Gynecology, Aretaieion Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
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26
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Ichikawa K, Shibahara H, Shirasuna K, Kuwayama T, Iwata H. Cell-free DNA content in follicular fluid: A marker for the developmental ability of porcine oocytes. Reprod Med Biol 2020; 19:95-103. [PMID: 31956291 PMCID: PMC6955585 DOI: 10.1002/rmb2.12309] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 11/01/2019] [Accepted: 11/26/2019] [Indexed: 01/18/2023] Open
Abstract
PURPOSE The present study examined the relationships among the amount of cell-free-DNA (cfDNA) in porcine follicular fluid (FF), the developmental ability of enclosed oocytes, and characteristics of granulosa cells and examined the effect of cfDNA content in maturation medium on the developmental ability of the oocytes. METHODS Oocytes and FF were collected from individual gilts, and the gilts were rated based on the ability of their oocytes to develop to the blastocyst stage and the amount of cfDNA in the FF. The copy numbers of mitochondrial DNA (Mt-DNA) and nuclear DNA (N-DNA) were measured by real-time PCR and the DNA sequence. FF or cfDNA was added to the maturation medium, and the developmental ability of the oocytes was examined. RESULTS The amount of cfDNA was associated with apoptosis of the granulosa cells, and high-cfDNA content in FF was associated with low developmental ability of oocytes. Supplementation of the maturation medium with FF containing high cf-Mt-DNA or with DNA extracted from the FF did not affect oocyte developmental competence. CONCLUSIONS Cell-free DNA content in FF is a marker for oocyte competence, but cfDNA in the oocyte maturation environment did not affect oocyte developmental ability.
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Affiliation(s)
- Kana Ichikawa
- Department of Animal ScienceTokyo University of AgricultureKanagawaJapan
| | - Hidenori Shibahara
- Department of Animal ScienceTokyo University of AgricultureKanagawaJapan
| | - Komei Shirasuna
- Department of Animal ScienceTokyo University of AgricultureKanagawaJapan
| | - Takehito Kuwayama
- Department of Animal ScienceTokyo University of AgricultureKanagawaJapan
| | - Hisataka Iwata
- Department of Animal ScienceTokyo University of AgricultureKanagawaJapan
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27
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Martinez RM, Baccarelli AA, Liang L, Dioni L, Mansur A, Adir M, Bollati V, Racowsky C, Hauser R, Machtinger R. Body mass index in relation to extracellular vesicle-linked microRNAs in human follicular fluid. Fertil Steril 2019; 112:387-396.e3. [PMID: 31146888 PMCID: PMC6663626 DOI: 10.1016/j.fertnstert.2019.04.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 03/28/2019] [Accepted: 04/01/2019] [Indexed: 12/17/2022]
Abstract
OBJECTIVE To study whether increased body mass index is associated with altered expression of extracellular vesicle microRNAs (EV-linked miRNAs) in human follicular fluid. DESIGN Cross-sectional study. SETTING Tertiary-care university-affiliated center. PATIENT(S) One hundred thirty-three women undergoing in vitro fertilization (IVF) were recruited from January 2014 to August 2016. INTERVENTIONS(S) None. MAIN OUTCOME MEASURE(S) EV-linked miRNAs were isolated from follicular fluid and their expression profiles were measured with the use of the Taqman Open Array Human miRNA panel. EV-linked miRNAs were globally normalized and inverse-normal transformed. Associations between body mass index (BMI) and EV-linked miRNA outcomes were analyzed by means of multivariate linear regression and principal component analysis. RESULT(S) Eighteen EV-linked miRNAs were associated with an increase in BMI after adjusting for age, ethnicity, smoking status, and batch effects. Hsa-miR-328 remained significant after false discovery rate adjustments. Principal component analyses identified the first principal component to account for 40% of the variation in our EV-linked miRNA dataset, and adjusted linear regression found that the first principal component was significantly associated with BMI after multiple testing adjustments. Using Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we predicted gene targets of EV-linked miRNA in silico and identified PI3K-Akt signaling, ECM-receptor interaction, focal adhesion, FoxO signaling, and oocyte meiosis pathways. CONCLUSION(S) These results show that a 1-unit increase in BMI is associated with altered follicular fluid expression of EV-linked miRNAs that may influence follicular and oocyte developmental pathways. Our findings provide potential insight into a mechanistic explanation for the reduced fertility rates associated with increased BMI.
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Affiliation(s)
- Rosie M Martinez
- Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts; Laboratory of Precision Environmental Biosciences, Department of Environmental Health Sciences, Columbia Mailman School of Public Health, New York, New York
| | - Andrea A Baccarelli
- Laboratory of Precision Environmental Biosciences, Department of Environmental Health Sciences, Columbia Mailman School of Public Health, New York, New York
| | - Liming Liang
- Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts
| | - Laura Dioni
- Epidemiology, Epigenetics, and Toxicology Laboratory, Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy
| | - Abdallah Mansur
- Department of Obstetrics and Gynecology, Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Ramat-Gan, Israel
| | - Michal Adir
- Department of Obstetrics and Gynecology, Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Ramat-Gan, Israel
| | - Valentina Bollati
- Epidemiology, Epigenetics, and Toxicology Laboratory, Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy
| | - Catherine Racowsky
- Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Russ Hauser
- Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts
| | - Ronit Machtinger
- Department of Obstetrics and Gynecology, Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Ramat-Gan, Israel.
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28
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Liu Y, Shen Q, Zhao X, Zou M, Shao S, Li J, Ren X, Zhang L. Cell-free mitochondrial DNA in human follicular fluid: a promising bio-marker of blastocyst developmental potential in women undergoing assisted reproductive technology. Reprod Biol Endocrinol 2019; 17:54. [PMID: 31291946 PMCID: PMC6621940 DOI: 10.1186/s12958-019-0495-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Accepted: 06/28/2019] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Cell-free mitochondrial DNA (cf-mtDNA) in body fluids has attracted much attention for the purpose of monitoring disease because of the clinical advantages. This study investigated whether the cf-mtDNA content in human follicular fluid samples was associated with oocyte and embryo developmental competence. METHODS We collected 225 individual follicular fluid samples from 92 patients undergoing conventional in vitro fertilization (n = 53) or intracytoplasmic sperm injection (n = 39). cf-mtDNA and cell-free nuclear DNA (cf-nDNA) were measured using real-time quantitative PCR for the ND1 and β-globin genes. Multivariate logistic regression and linear regression were used to analyze data. RESULTS The relative cf-mtDNA content (cf-ND1/cf-β-globin ratio) in follicular fluid was significantly lower in the group showing blastocyst development than in the non-blastocyst group (P = 0.030). Additionally, the relative cf-mtDNA content was significantly and positively correlated with the age of the female patient (P = 0.009), while the relative cf-mtDNA content for older women (≥38 years old) with anti-Müllerian hormone (AMH) ≤1.1 ng/ml was significantly higher than in those with AMH > 1.1 ng/ml (P <0.05). The cf-nDNA content was significantly positively correlated with the antral follicle count (P = 0.012), and significantly negatively correlated with both the number of days of stimulation and the total dose of gonadotropin administration (P = 0.039 and P = 0.015, respectively). Neither cf-mtDNA nor cf-nDNA levels in follicular fluid were associated with oocyte maturation, fertilization, or Day 3 embryo morphological scoring. CONCLUSIONS The relative cf-mtDNA content in human follicular fluid was negatively correlated with blastulation and positively correlated with the patient age, indicating that it is a promising bio-marker to evaluate oocyte developmental competence.
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Affiliation(s)
- Yu Liu
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China
| | - Qiuzi Shen
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China
| | - Xue Zhao
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China
| | - Min Zou
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China
| | - Shumin Shao
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China
| | - Jiao Li
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China
| | - Xinling Ren
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People's Republic of China
| | - Ling Zhang
- Family Planning Research Institute and Center for Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,, 430030, People's Republic of China.
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Adriaenssens T, Van Vaerenbergh I, Coucke W, Segers I, Verheyen G, Anckaert E, De Vos M, Smitz J. Cumulus-corona gene expression analysis combined with morphological embryo scoring in single embryo transfer cycles increases live birth after fresh transfer and decreases time to pregnancy. J Assist Reprod Genet 2019; 36:433-443. [PMID: 30627993 DOI: 10.1007/s10815-018-01398-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2018] [Accepted: 12/21/2018] [Indexed: 12/17/2022] Open
Abstract
PURPOSE Clinical pregnancy rate after IVF with eSET stagnates between 30 and 40%. In order to increase pregnancy and live birth rates, multiple embryo transfer is still common practice. Providing additional non-invasive tools to choose the competent embryo for transfer could avoid multiple pregnancy and improve time to pregnancy. Cumulus mRNA analysis with quantitative PCR (QPCR) is a non-invasive approach. However, so far, no gene sets have been validated in prospective interventional studies. METHODS A prospective interventional single-center pilot study with two matched controls (day-3 and day-5 eSET) was performed in 96 patients consenting to the analysis of the cumulus-corona of their oocytes. All patients were super-ovulated for ICSI and eSET at day 3. All oocytes were denuded individually and cumulus was analyzed by quantitative PCR using three predictive genes (EFNB2, SASH1, CAMK1D) and two housekeeping genes (UBC and β2M). Patients (n = 62) with 2 or more day-3 embryos (good or excellent morphology) had their embryo chosen following the normalized expression of the genes. RESULTS Corona testing significantly increased the clinical pregnancy and live births rates (63% and 55%) compared to single embryo transfer (eSET) on day 3 (27% and 23%: p < 0.001) and day 5 (43% and 39%: p = 0.022 and p = 0.050) fresh transfer cycle controls with morphology-only selection. Time-to-pregnancy was significantly reduced, regardless of the number of good-quality embryos available on day 3. CONCLUSION Combining standard morphology scoring and cumulus/corona gene expression analysis increases day-3 eSET results and significantly reduces the time to pregnancy. TRIAL REGISTRATION NUMBER This is not an RCT study and was only registered by the ethical committee of the University Hospital UZBRUSSEL of the Vrije Universiteit Brussel VUB (BUN: 143201318000).
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Affiliation(s)
- T Adriaenssens
- Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - I Van Vaerenbergh
- Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - W Coucke
- Department of Clinical Biology, Scientific Institute of Public Health, 1050, Brussels, Belgium
| | - I Segers
- Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, 1090, Brussels, Belgium
| | - G Verheyen
- Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, 1090, Brussels, Belgium
| | - E Anckaert
- Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - M De Vos
- Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, 1090, Brussels, Belgium
| | - J Smitz
- Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.
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Follicular fluid humanin concentration is related to ovarian reserve markers and clinical pregnancy after IVF-ICSI: a pilot study. Reprod Biomed Online 2018; 38:108-117. [PMID: 30503199 DOI: 10.1016/j.rbmo.2018.11.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2017] [Revised: 11/02/2018] [Accepted: 11/02/2018] [Indexed: 01/09/2023]
Abstract
RESEARCH QUESTION Is humanin present in the human ovary and follicular fluid? What relationship exists between humanin concentration in the follicular fluid and ovarian reserve and clinical outcomes after IVF and intracytoplasmic sperm injection (ICSI)? DESIGN Follicular fluid samples were collected from 179 patients undergoing their first IVF or ICSI cycle during oocyte retrieval. Ovarian tissues were collected from two patients undergoing surgery for ovarian cysts. Ovarian humanin localization was analysed using immunofluorescence staining. Expression of humanin in granulosa cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis. Follicular fluid humanin levels were evaluated with enzyme-linked immunosorbent assay. Relationships between follicular fluid humanin levels and ovarian reserve markers and clinical outcomes were analysed. RESULTS Strong humanin expression was found in the granulosa cells, oocytes and stromal cells of the ovary. Agarose gel electrophoresis of RT-PCR products showed rich humanin mRNA expression in human granulosa cells (119 bp). Follicular fluid humanin concentrations ranged from 86.40 to 417.60 pg/ml. They significantly correlated with FSH (r = -0.21; P < 0.01), LH (r = -0.18; P = 0.02), antral follicle count (r = 0.27; P < 0.01), anti-Müllerian hormone (r = 0.24; P = 0.03) and inhibin B (r = 0.46; P < 0.01) levels. Patients were subdivided into four groups according to follicular fluid humanin concentration quartiles (Q1-Q4). Patients in Q4 were more likely to achieve a pregnancy than Q1 (OR = 3.60; 95% CI 1.09 to 11.84). CONCLUSIONS Humanin concentration in the follicular fluid was positively associated with ovarian reserve and clinical pregnancy rate.
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Kansaku K, Munakata Y, Itami N, Shirasuna K, Kuwayama T, Iwata H. Mitochondrial dysfunction in cumulus-oocyte complexes increases cell-free mitochondrial DNA. J Reprod Dev 2018; 64:261-266. [PMID: 29618676 PMCID: PMC6021605 DOI: 10.1262/jrd.2018-012] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
This study examined the concentration of cell-free mitochondrial DNA (cf-mtDNA) in porcine follicular fluid (FF) and explored whether the cfDNA level in the culture medium could reflect mitochondrial dysfunction in cumulus cell-oocyte complexes (COCs). cfDNA concentration was higher in the fluid of small-sized follicles, compared to that in larger follicles. The length of cfDNA ranged from short (152 bp) to long (1,914 bp) mtDNA in FF, detected by polymerase chain reaction (PCR). cfDNA concentration in FF significantly correlated with the mtDNA copy number in FF but not with the number of one-copy gene (nuclear DNA) in FF. When the COCs were treated with the mitochondrial uncoupler, namely carbonyl cyanide m-chlorophenyl hydrazone (CCCP), for 2 h and incubated for 42 h, subsequent real-time PCR detected significantly higher amount of cf-mtDNA, compared to nuclear cfDNA, in the spent culture medium. The mtDNA number and viability of cumulus cells and oocytes remained unchanged. When the oocytes were denuded from the cumulus cells following CCCP treatment, PCR detected very low levels of cfDNA in the spent culture medium of the denuded oocytes. In contrast, CCCP treatment of granulosa cells significantly increased the amount of cf-mtDNA in the spent culture medium, without any effect on other markers, including survival rate, apoptosis of cumulus cells, and lactate dehydrogenase levels. Thus, cf-mtDNA was present in FF in a wide range of length, and mitochondrial dysfunction in COCs increased the active secretion of cf-mtDNA in the cultural milieu.
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Affiliation(s)
- Kazuki Kansaku
- Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
| | - Yasuhisa Munakata
- Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
| | - Nobuhiko Itami
- Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
| | - Koumei Shirasuna
- Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
| | - Takehito Kuwayama
- Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
| | - Hisataka Iwata
- Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
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Revelli A, Canosa S, Bergandi L, Skorokhod OA, Biasoni V, Carosso A, Bertagna A, Maule M, Aldieri E, D’Eufemia MD, Evangelista F, Colacurci N, Benedetto C. Oocyte polarized light microscopy, assay of specific follicular fluid metabolites, and gene expression in cumulus cells as different approaches to predict fertilization efficiency after ICSI. Reprod Biol Endocrinol 2017; 15:47. [PMID: 28645283 PMCID: PMC5481970 DOI: 10.1186/s12958-017-0265-2] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND The complex relationship between oocyte morphology, specific follicular fluid metabolites, gene expression in cumulus granulosa cells, and oocyte competence toward fertilization and embryo development still needs further clarification. METHODS Forty-six oocytes retrieved from the largest pre-ovulatory follicle of patients undergoing intra-cytoplasmic sperm injection (ICSI) were considered assessing: (a) oocyte morphological characteristics at polarized light microscopy (PLM), (b) specific follicular fluid (FF) metabolites previously suggested to influence oocyte competence (AMH, markers of redox status and of cytotoxicity), (c) transcription of AMH and AMH type II receptor genes in cumulus cells. Data were analyzed using mono-parametric tests and multivariable logistic analysis in order to correlate morphological and biochemical data with fertilization. RESULTS Comparing normally fertilized oocytes (n = 29, F group) with unfertilized (n = 17, nF group) we observed that: (a) the meiotic spindle area and major axis were significantly higher in nF group and in fertilized oocytes undergoing an early embryo development arrest; (b) AMH level in FF was comparable in F and nF groups; (c) the FF of nF group contained significantly higher levels of cytotoxicity (lactate dehydrogenase) and oxidative stress (Cu,Zn-superoxide dismutase, catalase, 4-hydroxynonenal-protein conjugates) markers; (d) cumulus cells of nF group showed significantly higher AMH receptor type II gene expression. CONCLUSIONS Taken together, these observations suggest that an excessive cytotoxicity level can alter AMH signal transduction within cumulus cells, in turn leading to partial inhibition of aromatase activity, altered cytoplasmic maturation and increased oxidative stress, factors able to impair oocyte fertilization competence and embryo growth.
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Affiliation(s)
- Alberto Revelli
- 0000 0001 2336 6580grid.7605.4Chair of Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, Department of Surgical Sciences, S. Anna Hospital, University of Torino, Torino, Italy
| | - Stefano Canosa
- 0000 0001 2336 6580grid.7605.4Chair of Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, Department of Surgical Sciences, S. Anna Hospital, University of Torino, Torino, Italy
| | - Loredana Bergandi
- 0000 0001 2336 6580grid.7605.4Department of Oncology, University of Torino, Torino, Italy
| | - Oleksii A. Skorokhod
- 0000 0001 2336 6580grid.7605.4Department of Oncology, University of Torino, Torino, Italy
| | - Valentina Biasoni
- 0000 0001 2336 6580grid.7605.4Chair of Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, Department of Surgical Sciences, S. Anna Hospital, University of Torino, Torino, Italy
| | - Andrea Carosso
- 0000 0001 2336 6580grid.7605.4Chair of Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, Department of Surgical Sciences, S. Anna Hospital, University of Torino, Torino, Italy
| | - Angela Bertagna
- 0000 0001 2336 6580grid.7605.4Endocrinology Laboratory, Department of Internal Medicine, University of Torino, Torino, Italy
| | - Milena Maule
- 0000 0001 2336 6580grid.7605.4Department of Medical Sciences, Cancer Epidemiology Unit, University of Torino, Torino, Italy
| | - Elisabetta Aldieri
- 0000 0001 2336 6580grid.7605.4Department of Oncology, University of Torino, Torino, Italy
| | - Maria Diletta D’Eufemia
- 0000 0001 2200 8888grid.9841.4Department of Woman, Child, General and Special Surgery, Second University of Napoli, Naples, Italy
| | - Francesca Evangelista
- 0000 0001 2336 6580grid.7605.4Chair of Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, Department of Surgical Sciences, S. Anna Hospital, University of Torino, Torino, Italy
| | - Nicola Colacurci
- 0000 0001 2200 8888grid.9841.4Department of Woman, Child, General and Special Surgery, Second University of Napoli, Naples, Italy
| | - Chiara Benedetto
- 0000 0001 2336 6580grid.7605.4Chair of Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, Department of Surgical Sciences, S. Anna Hospital, University of Torino, Torino, Italy
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Guan Y, Zhang W, Wang X, Cai P, Jia Q, Zhao W. Cell-free DNA induced apoptosis of granulosa cells by oxidative stress. Clin Chim Acta 2016; 473:213-217. [PMID: 27884753 DOI: 10.1016/j.cca.2016.11.023] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Revised: 11/14/2016] [Accepted: 11/14/2016] [Indexed: 11/17/2022]
Abstract
BACKGROUND Cell-free DNA is a DNA fragment that is produced by cell apoptosis which can affect the micro-environment of cell apoptosis. The levels of Cell-free DNA have been associated with successful rate of in vitro fertilization-embryo transfer (IVF-ET) and embryonic development. Our aim is to determine the relationship between cell-free DNA and embryo quality. The mechanisms of cell-free DNA in granulose and the apoptosis will be determined also. METHODS The study enrolled patients who were undergone IVF for the first time and grouped the patients as pregnant (n=130) and non-pregnant (n=59). The relationship was determined by statistical analysis between the levels of cell-free DNA in the follicular fluid and clinical data of IVF patients. Flow cytometry was done to detect the rate of granulosa cell apoptosis and intracellular reactive oxygen species (ROS) level. Western blotting and fluorescent quantitative PCR detected the apoptosis-related gene expressions. RESULTS Clinical data statistics showed that cell-free DNA levels were positively correlated with granulosa cell apoptosis and negatively correlated with embryo quality and pregnancy rates. High levels of cell-free DNA lead to increased ROS in granulosa cells and activated caspase through Fas/FasL that induced apoptosis. CONCLUSION High levels of cell-free DNA triggers granulosa cell apoptosis and influences oocyte maturation embryo development and pregnancy rates in IVF treatments. Cell-free DNA can be as a secondary criteria and predictive marker for the quality control of IVF embryo.
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Affiliation(s)
- Yichun Guan
- Reproduction Center, The Third Affiliated Hospital of ZhengZhou University, Henan, China
| | - Wenjuan Zhang
- Reproduction Center, The Third Affiliated Hospital of ZhengZhou University, Henan, China
| | - Xingling Wang
- Reproduction Center, The Third Affiliated Hospital of ZhengZhou University, Henan, China.
| | - Pengfei Cai
- Reproduction Center, The Third Affiliated Hospital of ZhengZhou University, Henan, China
| | - Qi Jia
- Reproduction Center, The Third Affiliated Hospital of ZhengZhou University, Henan, China
| | - Wenjie Zhao
- Reproduction Center, The Third Affiliated Hospital of ZhengZhou University, Henan, China
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Scalici E, Bechoua S, Astruc K, Duvillard L, Gautier T, Drouineaud V, Jimenez C, Hamamah S. Apolipoprotein B is regulated by gonadotropins and constitutes a predictive biomarker of IVF outcomes. Reprod Biol Endocrinol 2016; 14:28. [PMID: 27209151 PMCID: PMC4875704 DOI: 10.1186/s12958-016-0150-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Accepted: 03/18/2016] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND Follicular fluid (FF) is an important micro-environment influencing oocyte growth, its development competence, and embryo viability. The FF content analysis allows to identify new relevant biomarkers, which could be predictive of in vitro fertilization (IVF) outcomes. Inside ovarian follicle, the amount of FF components from granulosa cells (GC) secretion, could be regulated by gonadotropins, which play a major role in follicle development. METHODS This prospective study included 61 female undergoing IVF or Intra-cytoplasmic sperm injection (ICSI) procedure. Apolipoprotein B (APOB) concentrations in follicular fluid and APOB gene and protein expression in granulosa cells from reproductively aged women undergoing an in vitro fertilization program were measured. The statistical analyses were performed according to a quartile model based on the amount of APOB level found in FF. RESULTS Amounts of APOB were detected in human FF samples (mean ± SD: 244.6 ± 185.9 ng/ml). The odds of obtaining an oocyte in the follicle and a fertilized oocyte increased significantly when APOB level in FF was higher than 112 ng/ml [i.e., including in Quartile Q 2, Q3 and Q4] (p = 0.001; p < 0.001, respectively). The probabilities of obtaining an embryo and a top quality embryo on day 2, were significantly higher if APOB levels were within the ranges of 112 and 330 ng/ml (i.e. in Q2 and Q3) or 112 and 230 ng/ml (i.e. in Q2), respectively (p < 0.001; p = 0.047, respectively). In addition, our experiments in vitro indicated that APOB gene and protein expression, along with APOB content into culture were significantly under-expressed in GC upon stimulation with gonadotropins (follicular stimulating hormone: FSH and/or human chorionic gonadotropin: hCG). CONCLUSION We are reporting a positive and statistically significant associations between APOB and oocyte retrieval, oocyte fertilization, and embryo quality. Using an experimental study component, the authors report significant reduced APOB expression and content for luteinized granulosa cells cultured in the presence of gonadotropins.
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Affiliation(s)
- Elodie Scalici
- />ART-PGD Department, INSERM U1203, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
| | - Shaliha Bechoua
- />University of Burgundy, UFR Sciences de Santé de Dijon, Dijon, France
| | - Karine Astruc
- />Department of Epidemiology, Bocage Hospital, CHU Dijon, Dijon, France
| | | | - Thomas Gautier
- />INSERM UMR 866, lipids, Nutrition and Cancer, UFR of Medicine, Dijon, France
| | | | - Clément Jimenez
- />Service de Biologie de la Reproduction-CECOS, CHU de Bordeaux, Centre Aliénor d’Aquitaine, Bordeaux, France
| | - Samir Hamamah
- />ART-PGD Department, INSERM U1203, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
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Circulating microRNAs in follicular fluid, powerful tools to explore in vitro fertilization process. Sci Rep 2016; 6:24976. [PMID: 27102646 PMCID: PMC4840336 DOI: 10.1038/srep24976] [Citation(s) in RCA: 76] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Accepted: 04/08/2016] [Indexed: 12/19/2022] Open
Abstract
Circulating or “extracellular” microRNAs (miRNAs) detected in biological fluids, could be used as potential diagnostic and prognostic biomarkers of several disease, such as cancer, gynecological and pregnancy disorders. However, their contributions in female infertility and in vitro fertilization (IVF) remain unknown. This study investigated the expression profiles of five circulating miRNAs (let-7b, miR-29a, miR-30a, miR-140 and miR-320a) in human follicular fluid from 91 women with normal ovarian reserve and 30 with polycystic ovary syndrome (PCOS) and their ability to predict IVF outcomes. The combination of FF miR-30a, miR-140 and let-7b expression levels discriminated between PCOS and normal ovarian reserve with a specificity of 83.8% and a sensitivity of 70% (area under the ROC curve, AUC = 0.83 [0.73–0.92]; p < 0.0001). FF samples related to low number of mature oocytes (≤2) contained significant less miR-320a levels than those related to a number of mature oocytes >2 (p = 0.04). Moreover, FF let-7b predicted the development of expanded blastocysts with 70% sensitivity and 64.3% specificity (AUC = 0.67 [0.54–0.79]; p = 0.02) and FF miR-29a potential to predict clinical pregnancy outcome reached 0.68 [0.55–0.79] with a sensitivity of 83.3% and a specificity of 53.5% (p = 0.01). Therefore, these miRNAs could provide new helpful biomarkers to facilitate personalized medical care during IVF.
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Cell-free DNA and telomere length among women undergoing in vitro fertilization treatment. J Assist Reprod Genet 2015; 32:1697-703. [PMID: 26438644 DOI: 10.1007/s10815-015-0581-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2015] [Accepted: 09/22/2015] [Indexed: 10/23/2022] Open
Abstract
PURPOSE The current research is aimed at finding potential non-invasive bio-markers that will help us learn more about the mechanisms at play in failed assisted reproduction treatment. This exploratory pilot study examined the relationship between cell-free DNA (CFD) in plasma and telomere length in lymphocytes among women undergoing in vitro fertilization (IVF) and compared telomere length and CFD levels to a healthy control group. METHODS Blood of 20 women undergoing IVF was collected at three time points during the IVF cycle. We assessed the relationship between CFD and telomere length as well as controlling for morning cortisol levels. We also collected blood of 10 healthy controls at two time points (luteal and follicular phases of the menstrual cycle) and compared mean telomere length, CFD, and cortisol levels between the IVF patients and healthy controls. RESULTS The results revealed an inverse relationship between CFD levels and telomere lengths at several time points that remained significant even after controlling for cortisol levels. Women undergoing IVF had statistically significant higher levels of CFD and shorter telomeres compared to healthy controls. CONCLUSIONS The relationship between telomere length and CFD should be further explored in larger studies in order to uncover potential mechanisms that cause both shortened telomere length and elevated CFD in women undergoing IVF.
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[Circulating nucleic acids and infertility]. ACTA ACUST UNITED AC 2015; 43:593-8. [PMID: 26298813 DOI: 10.1016/j.gyobfe.2015.07.016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2015] [Accepted: 07/17/2015] [Indexed: 01/05/2023]
Abstract
Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART.
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Traver S, Scalici E, Mullet T, Molinari N, Vincens C, Anahory T, Hamamah S. Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes. PLoS One 2015; 10:e0136172. [PMID: 26288130 PMCID: PMC4545729 DOI: 10.1371/journal.pone.0136172] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Accepted: 07/30/2015] [Indexed: 12/27/2022] Open
Abstract
Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7–10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66–0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management.
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Affiliation(s)
- Sabine Traver
- CHU Montpellier, INSERM U1203, Saint-Eloi Hospital, Institute of Regenerative Medicine and Biotherapy, Montpellier, France
| | - Elodie Scalici
- CHU Montpellier, INSERM U1203, Saint-Eloi Hospital, Institute of Regenerative Medicine and Biotherapy, Montpellier, France
- Montpellier 1 University, UFR of Medicine, Montpellier, France
- ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
| | - Tiffany Mullet
- Montpellier 1 University, UFR of Medicine, Montpellier, France
- ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
| | | | - Claire Vincens
- ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
| | - Tal Anahory
- ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
| | - Samir Hamamah
- CHU Montpellier, INSERM U1203, Saint-Eloi Hospital, Institute of Regenerative Medicine and Biotherapy, Montpellier, France
- Montpellier 1 University, UFR of Medicine, Montpellier, France
- ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
- * E-mail:
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Scalici E, Hamamah S. Reply: Cell-free nucleic acids as non-invasive biomarkers of gynecological disorders, fetal aneuploidy and constitutional maternal chromosomal mosaicism. Hum Reprod Update 2015; 21:692. [PMID: 25765865 DOI: 10.1093/humupd/dmv014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Elodie Scalici
- CHU Montpellier, Institute of Regenerative Medicine and Biotherapy, Saint-Eloi Hospital, INSERM U1203, Montpellier, France Montpellier 1 University, UFR of Medicine, Montpellier, France ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
| | - Samir Hamamah
- CHU Montpellier, Institute of Regenerative Medicine and Biotherapy, Saint-Eloi Hospital, INSERM U1203, Montpellier, France Montpellier 1 University, UFR of Medicine, Montpellier, France ART-PGD Department, Arnaud de Villeneuve Hospital, CHU Montpellier, Montpellier, France
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