Regulation of Reproduction by Dopamine
Nira Ben-Jonathan in Dopamine, 2020
The resumption of meiosis (oocyte maturation) is triggered by the preovulatory LH surge. The meiotic block is removed and the primary oocyte completes the first meiotic division. An asymmetric cytoplasmic division generates a large secondary oocyte and a small discarded cell, called the polar body. The secondary oocyte then enters meiosis II and becomes arrested at metaphase II. The resumption of meiosis at the time of ovulation is characterized by the dissolution of the nucleus, commonly referred to as germinal vesicle breakdown (GVBD). After GVBD, the chromatin is condensed into chromosomes, microtubules are organized into meiotic spindles, and the homologous chromosomes are separated to emit the first polar body. The post-GVBD maturational processes are controlled by histone modifications, centrosome proteins, various protein kinases/phosphatases, spindle checkpoints, and cytoskeletons.
Preimplantation genetic diagnosis for infertility
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
As well as advances in genetic testing, improvements to embryological practice have also contributed to the evolution of PGS. The development of blastocyst culture and trophectoderm biopsy have been particularly beneficial. Culturing the embryo to the blastocyst stage on day 5 selects out non-competent embryos, many of which arrest at earlier developmental stages (36). As a result, blastocyst culture is advantageous for IVF outcomes even without genetic testing (53). Blastocyst biopsy involves breach of the zona pellucida with a laser on day 3 or immediately prior to the procedure on days 5/6. Trophectoderm cells either herniate through the hole spontaneously or are actively drawn through it by suction. Three to ten of these cells (typically about five cells) can then be removed. The inner cell mass is not disturbed and therefore the technique does not affect cells that develop into the fetus. Trophectoderm biopsy is considered to pose significantly less risk to the embryo than cleavage-stage biopsy (37). Also, because several cells are taken, genetic tests tend to yield more robust data. Using some methods, especially NGS, mosaicism can be detected within the biopsy specimen. Blastocyst biopsy is increasingly becoming the first choice of biopsy method for PGS. Polar body biopsy is still currently used, but mostly in countries where genetic testing of the embryo itself is not permitted.
Pre-implantation genetic diagnosis
Sheila A.M. McLean, Sarah Elliston in Regulating Pre-Implantation Genetic Diagnosis, 2012
A single cell of the eight or so to which the embryo will have cleaved by day 3 in vitro, is easily accessible and removable after a hole is made in the shell (zona pellucida) which surrounds each embryo. This can be done by using an acidified medium or a laser. Early evidence suggests that the loss of a blastomere does not harm the embryo, as its further development is still plastic and uncommitted. However, the removal of more than one blastomere may be detrimental to the establishment of a pregnancy.18 Removal of a portion of the trophectoderm as a biopsy on day 5 has the practical advantage that fewer embryos will be available, as not all fertilised eggs progress to this stage, thus reducing the load on the laboratory. Equally, each of these embryos has better developmental potential to have reached this stage in vitro. The disadvantage is that the diagnosis will either have to be made within 24 hours in order to enable use to be made of the limited implantation window (by day 6), or the embryo will need to be frozen for subsequent replacement if that time interval cannot be met.19 Removal of the first polar body (and preferably also the second polar body) is easily effected at the time of fertilisation, and will reveal information from the maternally transmitted genome.20 It can also reveal important transmissible meiotic chromosome errors that account for the majority of sporadic aneuploidies.21
The role of ORC4 in enucleation of Murine Erythroleukemia (MEL) cells is similar to that in oocyte polar body extrusion
Published in Systems Biology in Reproductive Medicine, 2020
Hieu Nguyen, Anna Ung, W. Steven Ward
We were previously able to demonstrate that in murine oogenesis, the formation of the ORC4 cage was required for polar body extrusion. We tested whether ORC4 synthesis was required for the progression of MEL cells from exponentially growing to enucleating cells. MEL cells were transfected with plasmids containing expression vectors for either control or ORC4 siRNA, and then induced to progress to enucleation with vacuolin-1. Cells were stained for ORC4, and the four types of cells were counted after 48 hrs. Control MEL cells that were not treated with vacuolin-1 maintained an approximately 1 to 2.5 ratio of Type A to Type B cells (Figure 5). MEL cells that were treated with control siRNA that contained a scrambled sequence progressed to enucleated cells just as cells with no siRNA with vacuolin-1 did (Figure 5). However, when cells were transfected with siRNA directed against Orc4 mRNA and then treated with vacuolin-1 for 48 h, no enucleated cells could be detected. Moreover, the ratio of Type A to Type B was closer to 1 to 1, suggesting that even the progression from Type A, with no cytoplasmic ORC4 to Type B, with a low level of cytoplasmic ORC4, was decreased. Quantitative RT-PCR of the MEL cells treated with control versus Orc4-directed siRNA showed a significant decrease in Orc4 mRNA, but not a complete inhibition of Orc4 transcription (Figure 6).
Improvement of embryonic development and clinical outcomes of germinal vesicle stage oocytes using a microvibration culture system
Published in Systems Biology in Reproductive Medicine, 2019
Seong-Ho Yang, San-Hyun Yoon, Jae-Hoon Jung, Jin-Ho Lim, Yong Ko
The nuclear maturity of the retrieved oocytes was assessed using the dissecting microscope with high magnification (×80) using the sliding method (Chian et al. 2000). To exactly observe GV-stage oocytes, cumulus cells were partly removed using 25 IU hyaluronidase (MRC#hyase; Maria Fertility Hospital, Seoul, Korea) (Yoon et al. 2011) and mechanical pipetting. The GV-stage oocytes were transferred into an organ culture dish (60 × 15 mm, Falcon; Franklin Lakes, NJ, USA) containing 1 ml of IVM medium (YS medium; Maria Fertility Hospital, Seoul, Korea) (Yoon et al. 2001) supplemented with 30% of the patients’ own serum (inactivated at 56°C for 30 min) with a final concentration of 0.6 IU/ml recombinant human follicle-stimulating hormone (Gonal-F; Merck Serono, Geneva, Switzerland), 0.1 IU/ml HCG (LG), and 10 ng/ml recombinant human epidermal growth factor (Invitrogen, Seoul, Korea) at 37°C in 6% CO2, 5% O2, and 89% N2 with 95% humidity. After approximately 30 ~ 32 h of incubation in IVM medium, all COCs were denuded of the cumulus cells using 25 IU hyaluronidase and mechanical pipetting. Oocyte maturation was assessed by the presence of the first polar body in the perivitelline space.
The effects of letrozole on women with endometriosis undergoing ovarian stimulation for in vitro fertilization
Published in Gynecological Endocrinology, 2020
Se Jeong Kim, Chang Woo Choo, Seul Ki Kim, Jung Ryeol Lee, Byung Chul Jee, Chang Suk Suh, Won Don Lee, Seok Hyun Kim
All patients underwent COS using gonadotropin-releasing hormone (GnRH) agonist or antagonist for pituitary suppression. For patients receiving the combination therapy, 5 mg letrozole was started from the third day of the menstrual cycle and continued until the day of triggering. The recombinant FSH (Gonal-F; Serono, Geneva, Switzerland), purified human menopausal gonadotropin (Menopur; Ferring Pharmaceuticals Inc., Kiel, Germany), or recombinant FSH plus recombinant LH (Pergoveris; Serono, Darmstadt, Germany) were initiated with the starting dose based on the age and ovarian reserve. When the leading follicle reached a mean diameter of 14 mm, GnRH antagonist (Cetrorelix, 0.25 mg; Serono, Darmstadt, Germany) was added. Recombinant human chorionic gonadotropin (hCG; Ovidrel, 250 μg; Serono, Darmstadt, Germany) was given for final oocyte maturation when at least two follicles reached a mean diameter of 18 mm. About 34–36 h after hCG triggering, the oocyte was retrieved under ultrasound guidance. Fertilization was assessed by the presence of 2pronuclei (2PN) and a second polar body. Usable embryos were defined as transferred embryos plus cryopreserved embryos for future use. Fresh embryo transfers were mostly performed 3 days following retrieval. Vaginal progesterone was given for luteal support.