Paraffin oil

Manufactured by Vitrolife

Sourced in Sweden

Paraffin oil is a transparent, colorless liquid used in various laboratory applications. It serves as an inert overlay for in vitro fertilization (IVF) procedures, where it helps to maintain the optimal environment for embryo culture and development.

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Lab products found in correlation

Sequential medium, by Vitrolife (2 mentions) Cetrotide, by Merck Group (1 mentions) Ovidrel, by Merck Group (1 mentions) Embryoscope, by Vitrolife (1 mentions) Human recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions) G1 plus medium, by Vitrolife (1 mentions) Hyaluronidase, by Vitrolife (1 mentions) Spermgrad, by Vitrolife (1 mentions) G ivf plus, by Vitrolife (1 mentions) G 1 plus, by Vitrolife (1 mentions) Mco 5m, by Sanyo (1 mentions)

7 protocols using paraffin oil

Oocyte Retrieval and Fertilization Protocol

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All women were treated with either a gonadotropin-releasing hormone
(GnRH) agonist long protocol or a GnRH antagonist (Cetrotide, Merck Serono, Roma, Italy) protocol. Human chorionic gonadotropin (hCG; Ovidrel, Merck Serono) was given to the patients 36 hours prior to oocyte retrieval. Oocytes were retrieved via transvaginal ultrasound guidance with a 19-gauge needle (Dukwoo Medical, Hwaseong, Korea). Mature oocytes were inseminated with either ICSI or conventional insemination according to the state of the spermatozoa and oocytes, as well as the patient's previous IVF history [10 (link)]. The presence of two pronuclei was observed at 17 to 19 hours after insemination. Normal fertilized zygotes were cultured in 30-μL micro-drops in either a sequential medium (Vitrolife, Gothenburg, Sweden) or a 1-step medium (Life Global, Belgium) and overlaid with paraffin oil (Vitrolife) in an atmosphere of 6% CO₂, 5% O₂, and 95% humidity at 37℃.

Lee J.W., Cha J.H., Shin S.H., Kim Y.J., Lee S.K., Park C.K., Pak K.A., Yoon J.S, & Park S.Y. (2017). Efficacy of embryo transfer on day 2 versus day 3 according to maternal age in patients with normal ovarian response. Clinical and Experimental Reproductive Medicine, 44(3), 141-145.

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Embryo Culture with Optimized Conditions

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The TL culture system, EmbryoScope+ (Vitrolife, Kungsbacka, Sweden), was used in this study. The cultivation condition consisting of an atmosphere of 5% O₂, 5% CO₂, and 90% N₂ at 37°C was applied for oocytes and embryos. The media covered with paraffin oil (Vitrolife) were equilibrated in an incubator for at least 2 h before use, including the fertilization medium (SAGE Biopharma, Bedminster, NJ, United States) with 15% serum protein substitute (SPS; SAGE Biopharma) for conventional insemination or intracytoplasmic sperm injection (ICSI), cleavage medium (SAGE Biopharma) with 15% SPS, and blastocyst medium (SAGE Biopharma) with 15% SPS for embryo culture. On the basis of the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen, abnormal sperm quality was defined if at least one of the following properties was found: oligospermia (sperm concentration ≤15 × 10⁶/ml), asthenospermia (total motility ≤40%), and teratospermia (normal sperm form ≤4%). ICSI was used for all of the mature oocytes from couples with oligoasthenoteratozoospermia (OAT) or for approximately half of the mature oocytes from couples with non-OAT.

Chen C.H., Lee C.I., Huang C.C., Chen H.H., Ho S.T., Cheng E.H., Lin P., Chen C.I., Lee T.H, & Lee M.S. (2021). Blastocyst Morphology Based on Uniform Time-Point Assessments is Correlated With Mosaic Levels in Embryos. Frontiers in Genetics, 12, 783826.

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Oocyte Maturation and ICSI Fertilization Protocol

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The mature oocytes were inseminated 2 or 3 hours later by intracytoplasmic sperm injection (ICSI). The immature oocytes at Metaphase-I (MI) and germinal vesicle (GV) stages (Figure 1) were cultured in 1 mL maturation medium to induce final oocyte maturation at 37°C in 6% CO₂, 5% O₂ and 89% N₂ with high humidity. The IVM culture medium contained 30% serum of the patient’s own (inactivated at 56° for 30 min) with 75 mIU/mL follicle stimulating hormone (FSH, Serono, Switzerland), 10 mIU/ml human menopausal gonadotrophin (Livzon Medical Groups; Zhu Hai, China) and 10 ng/ml recombinant human epidermal growth factor (Invitrogen, Carlsbad, CA, USA). The maturity of MI stage oocytes was re-evaluated after 3 hours IVM culture and the matured oocytes were subjected to ICSI 2 hours later. After 24 to 48 hours of IVM, if the oocytes mature, they were then inseminated by ICSI.
Following 16–18 h after ICSI, fertilization was evaluated by the appearance of 2 distinct pronuclei and 2 polar bodies. The zygotes were cultured in 20 μL droplets of G1-PLUS medium (Vitrolife, Gothenburg, Sweden) covered with paraffin oil (Vitrolife) and incubated according to standard procedures for further development.

Li J.H., Sun T.C., Zhang S.W., Jiao T.T., Cheng Y.B., Dong P., Chian R.C, & Xu Y. (2022). Effect of dominant follicle status at the time of retrieval on the clinical outcomes in natural cycle IVF combined with immature oocyte treatment. Aging (Albany NY), 14(11), 4728-4738.

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Oocyte Retrieval and Sperm Preparation for ICSI

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The cumulus oocyte complexes (COCs) were retrieved after 36-h hCG injection under the guidance of transvaginal B-ultrasound. The retrieved COCs were immediately put into G-IVF Plus (Vitrolife Sweden AB, Goteborg, Sweden) medium and cultured for 1-2 h at 37°C and in the presence of 6% CO₂. Spermgrad (Vitrolife) at concentrations of 90% and 45% was used for density gradient centrifugation and G-IVF Plus for washing the ejaculated sperm. The testicular tissues from patients undergoing testicular sperm aspiration were shredded in G-IVF Plus solution with a 1-mL syringe needle, centrifuged, and washed directly. The granulosa cells around COCs were removed by repetitive aspiration with a 150-μm-diameter fine needle (Sunlight, FL, USA) with hyaluronidase (Vitrolife) digestion. Then, the oocytes removed from the granulosa cells were cultured in G-IVF Plus for 1–2 h for ICSI. After injection, the oocytes were cultured in a 50-μL G-1 Plus (Vitrolife) droplet covered with paraffin oil (Vitrolife) and cultured at 37°C and in the presence of 6% CO₂.

Xu Z., Yao G., Niu W., Fan H., Ma X., Shi S., Jin H., Song W, & Sun Y. (2021). Calcium Ionophore (A23187) Rescues the Activation of Unfertilized Oocytes After Intracytoplasmic Sperm Injection and Chromosome Analysis of Blastocyst After Activation. Frontiers in Endocrinology, 12, 692082.

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Trophectoderm Biopsy of Expanded Blastocysts

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TE biopsy was performed on day 5 or 6 expanded blastocysts. The blastocysts for TE biopsy were loaded in culture dishes, which contained two to three microdroplets of a blastocyst medium (Sage BioPharma, Inc.) overlaid with paraffin oil (Vitrolife, Kungsbacka, Sweden). The blastocysts were held using a holding pipette (Humagen, Charlottesville, VA, USA), and laser pulses were used to punch a small hole in the ZP away from the inner cell mass to accommodate the passage of several TE cells. Approximately 5–10 TE cells detached from the ZP were aspirated into the biopsy pipette with smooth suction. The aspirated cells were detached from the blastocysts with several laser pulses combined with smooth suction. The detached cells were aspirated into the TE biopsy pipette and released into the biopsy drop. The biopsied TE cells were immediately placed in RNAse—DNAse-free polymerase chain reaction tubes. The blastocyst morphology following TE biopsy was typically collapsed.

Chen H.H., Huang C.C., Cheng E.H., Lee T.H., Chien L.F, & Lee M.S. (2017). Optimal timing of blastocyst vitrification after trophectoderm biopsy for preimplantation genetic screening. PLoS ONE, 12(10), e0185747.

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Fertilization: Conventional vs. ICSI

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Mature oocytes were inseminated with either conventional insemination or intracytoplasmic sperm injection (ICSI), depending on the state of sperm, oocyte and previous history of patient, etc. The outcome of fertilization was assessed in 17-19 hours after insemination, when the presence of two pronuclei was observed. Up to three zygotes were cultured in 30 μL of micro-drop with either one-step medium (Life Global^®, Belgium) or sequential medium (Vitrolife, Sweden) under covering with paraffin oil (Vitrolife, Sweden) in the atmosphere of 6% CO₂, 5% O₂ and 95% humidity at 37°C.

Lee J.W., Cha J.H., Shin S.H., Kim Y.J., Lee S.K., Cha H.J., Kim J.H., Ahn J.H., Kim H.Y., Pak K.A., Yoon J.S., Park S.Y, & Park C.K. (2016). Long Cut Straw Provides Stable the Rates of Survival, Pregnancy and Live Birth for Vitrification of Human Blasotcysts. Development & Reproduction, 20(3), 219-225.

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Embryo Culture and Evaluation for IVF

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For IVF procedures, oocytes were inseminated with motile spermatozoa at a concentration of 10,000 per ml. For ICSI, oocytes were microinjected with sperm 4–6 h after oocyte retrieval. The fertilized oocytes were transferred into pre-equilibrated culture dishes (Thermo Scientific, Waltham, MA, USA) with 25 μl of culture medium (Vitrolife Sweden, Gothenburg, Sweden) covered with 1.2 ml of paraffin oil (Vitrolife Sweden). The embryos were cultured in an incubator (MCO-5 M, Sanyo, Osaka, Japan) at 37 °C with 5% O₂ and 6% CO₂ until ET on day 3.
Embryo morphology was assessed according to the guidelines of the European Society of Human Reproduction and Embryology/Alpha consensus [50 (link)]. Embryos were graded according to blastomere number, blastomere size, and degree of fragmentation, using a morphological scoring system, accounting for the regularity of the blastomeres, degree of fragmentation, and microscopic appearance of the embryos. Transferable embryos were defined by the presence of 6–10 symmetrical blastomeres and < 20% fragmentation, with no multinucleation, on day 3.

Li J., Chen Q., Wang J., Huang G, & Ye H. (2020). Does growth hormone supplementation improve oocyte competence and IVF outcomes in patients with poor embryonic development? A randomized controlled trial. BMC Pregnancy and Childbirth, 20, 310.

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