A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm v2

Author(s):  
Magdalena Wigger ◽  
Simon E Tröder ◽  
Branko Zevnik

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. This protocol is compatible with frozen sperm from all major repositories and the IVF can easily be adapted to accommodate freshly harvested sperm.

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0259202
Author(s):  
Magdalena Wigger ◽  
Simon E. Tröder ◽  
Branko Zevnik

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.


2015 ◽  
Vol 33 (22) ◽  
pp. 2424-2429 ◽  
Author(s):  
Kutluk Oktay ◽  
Volkan Turan ◽  
Giuliano Bedoschi ◽  
Fernanda S. Pacheco ◽  
Fred Moy

Purpose We have previously reported an approach to ovarian stimulation for the purpose of fertility preservation (FP) in women with breast cancer via embryo freezing with the concurrent use of letrozole. The aim of this study was to provide the pregnancy and FP outcomes when embryos generated with the same protocol are used. Patients and Methods In all, 131 women with stage ≤ 3 breast cancer underwent ovarian stimulation and received concurrent letrozole 5 mg per day before receiving adjuvant chemotherapy and cryopreserving embryos. Results Thirty-three of the 131 women underwent 40 attempts to transfer embryos to their own uterus (n = 18) or via the use of a gestational carrier (n = 22) at a mean age of 41.5 ± 4.3 years with a median 5.25 years after embryo cryopreservation. The overall live birth rate per embryo transfer was similar to the US national mean among infertile women of a similar age undergoing in vitro fertilization–embryo transfer (45.0 v 38.2; P = .2). Seven (38.8%) of the 18 pregnancies were twins with no higher-order pregnancies being encountered. No fetal anomalies or malformations were reported in 25 children after a mean follow-up of 40.4 ± 26.4 months. Seventeen of the 33 women attempting pregnancy had at least one child, translating into an FP rate of 51.5% per attempting woman. Conclusion Embryo cryopreservation after ovarian stimulation with the letrozole and follicle-stimulating hormone protocol preserves fertility in women with breast cancer and results in pregnancy rates comparable to those expected in a noncancer population undergoing in vitro fertilization.


2012 ◽  
Vol 61 (1) ◽  
Author(s):  
José María Pardo Sáenz

Lo scopo di questo articolo è quello di mostrare il volto sconosciuto dei metodi artificiali di riproduzione umana. In generale, le cliniche per la fecondazione umana mostrano solo il volto più attraente della fecondazione in vitro: il neonato. Eppure, non hanno mai mostrato l’altra faccia di essa: la procedura lunga e drammatica che sta dietro. Dopo aver fatto un breve cenno alla attribuzione del Premio Nobel per la Medicina 2010 Robert Edwards, padre della fecondazione in vitro, analizziamo le due radici che sostengono il grande albero di metodi artificiali di riproduzione umana: lo scientismo e il sentimentalismo. Poi si studia l’aspetto etico di queste procedure. Siamo giunti ad un giudizio eticamente negativo su queste tecniche, dal momento che snaturano il significato della sessualità umana e l’atto coniugale. Subito dopo, analizziamo alcune delle conseguenze più frequenti di tali metodi: “la mortalità embrionale” associata con tali tecniche; “il congelamento degli embrioni in eccesso”; “i rischi per la salute”, sia del neonato così come quella della donna. Tutti questi sono elementi che la coppia deve prendere in considerazione al fine di prendere la decisione più libera e più responsabile possibile. Infine, spieghiamo il “pendio scivoloso” relativo all’uso di queste tecniche (il bambino medicalizzato, i bambini à la carte, la maternità surrogata e il mercantilismo riproduttivo): ciò che è stato sviluppato con uno intento ben preciso, per risolvere il problema di infertilità, ha sicuramente aperto nuove importanti questioni etiche. ---------- The purpose of this article is to show the unknown face of artificial human reproduction methods. Generally, human fertilization clinics only show the most attractive face of in vitro fertilization: the newborn. Yet, they never show the other face of it: the long and dramatic procedure behind it. After briefly mentioning the 2010 Nobel Medicine Award granted to Robert Edwards, the father of in vitro fertilization, we analyze the two roots that support the big tree of artificial human reproduction methods: scientism and sentimentalism. Then we study the ethical aspect of these procedures. We come to the conclusion that these techniques are ethically negative since they denaturalize the meaning of human sexuality and the conjugal act. Right after that, we analyze some of the most frequent consequences of such methods: “embryonic mortality” and directly related to it exceeding “embryo freezing”; “the risks for the health”, both of the newborn as well as that of the woman. All those are elements that the couple must take into consideration in order to make the freest and most responsible decision possible. Finally, we explain the “slippery slope” effect in the use of these techniques (medicine babies, babies a la carte, surrogate maternity and reproductive mercantilism): what was developed with a very specific aim in mind, to remedy the infertility problem, has definitely open new and important ethical concerns.


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