A Review on In-vitro Haploid Production in Orchids

The aim of writing this paper is to review production of haploids in orchids in vitro. Haploids possess half number of chromosomes and do not undergo fertilization. In vitro conditions provide necessary nutrients and conditions that are required for growth of haploid plantlets. The natural breeding cycle of orchids is very slow as well as unpredictable. To reduce this time, the technique of in vitro haploid production is used. In vitro conditions can decrease or shorten the time required for juvenile period in Orchids. Haploid plants are produced from in vitro haploid culture. This technique is useful to produce homozygous pure lines and to increase the yield of a plant.

A Review on Microfluidics: An Aid to Assisted Reproductive Technology

Infertility is a state of the male or female reproductive system that is defined as the failure to achieve pregnancy even after 12 or more months of regular unprotected sexual intercourse. Assisted reproductive technology (ART) plays a crucial role in addressing infertility. Various ART are now available for infertile couples. Fertilization in vitro (IVF), intracytoplasmic sperm injection (ICSI) and intrauterine insemination (IUI) are the most common techniques in this regard. Various microfluidic technologies can incorporate various ART procedures such as embryo and gamete (sperm and oocyte) analysis, sorting, manipulation, culture and monitoring. Hence, this review intends to summarize the current knowledge about the application of this approach towards cell biology to enhance ART.

Influence of conception and delivery mode on stress response marker Oct4B1 and imprinted gene expression related to embryo development: A cohort study

Background: Recent scientific data support that the mode of conception and delivery may influence epigenetic regulation and therefore embryo development. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel variant of OCT4 with yet unknown biological function, is suggested to have a potential role in mediating cellular stress response. Furthermore, Insulinlike Growth Factor 2 (IGF2), Mesoderm-specific Transcript (MEST) and paternally expressed gene 10 (PEG10) are genes known as imprinted and are regulated via means of epigenetic regulation. The influence of delivery mode and conception on epigenetic regulation is an active research field. Objective: Our aim was to correlate the expression level of Oct4B1 and the expression and methylation level of IGF2, MEST, and PEG10 imprinted genes with the mode of delivery and conception in the umbilical cord blood of newborns. Materials and Methods: Samples of umbilical cord blood from infants born after vaginal delivery, caesarean section (CS) with the infant in cephalic position and CS due to breech position were examined. Furthermore, the investigation included infants conceived through means of assisted reproductive technology. Results: No statistically significant differences were found in mRNA expression levels between different modes of conception and delivery (p = 0.96). Oct4B1, IGF2, MEST, and PEG10 expression levels do not seem to be significantly affected by different modes of conception and delivery. Conclusion: These results indicate that the expression and methylation patterns of Oct4B1, IGF2, MEST and PEG10 in umbilical cord blood are not affected by the conception and delivery mode. Key words: Conception, Fertilization in vitro, Genomic imprinting, Fetal blood.

Complicações Gestacionais Relacionadas À Fertilização In Vitro

Introdução: A indicação da fertilização in vitro (FIV) se faz necessária diante da impossibilidade de reprodução por outras maneiras ou, ainda, em casos de mulher com idade superior a 35 anos, história familiar de menopausa precoce e realização de radioterapia na região pélvica. O objetivo deste artigo foi descrever e enfatizar as principais complicações em gestantes, relacionadas ao processo de FIV, garantindo a informação sobre resultados adversos da gravidez, possibilitando, então, o diagnóstico e tratamento dessas complicações precocemente. Método: Trata-se de uma revisão bibliográfica do tipo integrativa, com abordagem qualitativa, de procedimento bibliográfico, natureza aplicada e objetivo descritivo de estudos nacionais e internacionais. Tal pesquisa foi realizada por meio da base de dados PubMed e seus sites aliados, no período de novembro de 2020 à janeiro de 2021, sendo: “Pregnancy complications”, “Related” e”Fertilization in vitro” os descritores usados. Diante disso, foram obtidos 19 artigos no total. Resultados: Foram avaliados 10 artigos, sendo estudos transversais e de coorte, os mais presentes. Dentre os estudos analisados, 40% (n=4) apontaram sobre o impacto em complicações gestacionais, 30% (n=3) demonstraram que há um impacto da variante psicológica das gestantes e, em 20% (n=2), avaliou-se os mecanismos de TEV (Tromboembolismo Venoso), associando as gestações à FIV com aumento do tromboembolismo. Conclusão: A análise dos estudos relacionados à FIV confirma que esse é um dos métodos recorridos como opção para concepção, quando essa não pode ser feita de outras formas. No entanto, embora haja dúvidas acerca dos riscos, se comparados aos de uma gestação natural, pode haver limitações e complicações relacionadas ao método. Dentre os estudos avaliados a respeito do impacto em complicações gestacionais, concluiu-se que gestações em FIV apresentam maiores riscos se comparadas à gestação natural. Dado todo o exposto, torna-se notória a necessidade de conhecimento sobre as possíveis complicações e uma adequada análise e indicação do método FIV, a fim de evitar ou identificar e tratar precocemente tais complicações e alcançar os benefícios desejados.

Follicular Fluid from Infertile Women with Mild Endometriosis Impairs In Vitro Bovine Embryo Development: Potential Role of Oxidative Stress

Objective To investigate whether follicular fluid (FF) from infertile women with mild endometriosis (ME) alters in vitro bovine embryo development, and whether the antioxidants N-acetyl-cysteine (NAC) and/or L-carnitine (LC) could prevent such damages. Methods Follicular fluid was obtained from infertile women (11 with ME and 11 control). Bovine oocytes were matured in vitro divided in: No-FF, with 1% of FF from control women (CFF) or ME women (MEFF); with 1.5 mM NAC (CFF + NAC, MEFF + NAC), with 0.6 mg/mL LC (CFF + LC, MEFF + LC), or both antioxidants (CFF + NAC + LC, MEFF + NAC + LC). After in vitro fertilization, in vitro embryo culture was performed for 9 days. Results A total of 883 presumptive zygotes were cultured in vitro. No differences were observed in cleavage rate (p = 0.5376) and blastocyst formation rate (p = 0.4249). However, the MEFF group (12.5%) had lower hatching rate than the No-FF (42.1%, p = 0.029) and CFF (42.9%, p = 0.036) groups. Addition of antioxidants in the group with CFF did not alter hatching rate (p ≥ 0.56), and in groups with MEFF, just NAC increased the hatching rate [(MEFF: 12.5% versus MEFF + NAC: 44.4% (p = 0.02); vs MEFF + LC: 18.8% (p = 0.79); versus MEFF + NAC + LC: 30.8% (p = 0.22)]. Conclusion Therefore, FF from infertile women with ME added to medium of in vitro maturation of bovine oocytes impairs hatching rate, and NAC prevented these damages, suggesting involvement of oxidative stress in worst of oocyte and embryo quality of women with ME.

The effect of the human cumulus cells-conditioned medium on in vitro maturation of mouse oocyte: An experimental study

Background: To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell culture methods, one of the approaches to improve this technology is conditioned medium from different sources. It is desirable to apply in vitro maturation (IVM) and use oocytes from normal cycles instead of stimulating ovulation. Objective: To investigate the effect of human cumulus cell condition medium (hCCCM) on the IVM of immature mouse oocytes and morphology. Materials and Methods: In this experimental study, 240 germinal vesile oocytes were collected from four-six wk-old mice after 48 hr of 5IU pregnant mare serum gonadotropin (PMSG) injection and cultured in hCCCM (test group, n = 120) and DMEM + 20% FBS (control group, n = 120). The IVM rates and changes in perivitelline space (PVS) and shape were investigated at 8, 16, and 24 hr following the culture. The mature (MII) oocytes were subjected to in vitro fertilization (IVF) and the fertilization rate was assessed in three days. Results: A significant difference was observed between the maturation rates in the hCCCM and control groups (24.16% vs 0%; p = 0.001), as well as morphologic changes between the two groups (p = 0.04, p = 0.05). The development rate for MII oocytes attained from IVM in the hCCCM group was 27.58% (2-cell) and 6.89% (4-cell). Data displayed that hCCCM is an effective medium for oocytes maturation compared to the control medium. Conclusion: hCCCM supports oocyte in vitro growth and maturation. Moreover, hCCCM changes the oocyte shape and size of perivitelline space. Key words: Germinal vesicle, Cumulus cell, Conditioned medium, In vitro fertilization, In vitro maturation, Oocyte.

Using a Dielectrophoretic Microfluidic Biochip Enhanced Fertilization of Mouse Embryo in Vitro

Droplet microfluidics has appealed to many interests for its capability to epitomize cells in a microscale environment and it is also a forceful technique for high-throughput single-cell epitomization. A dielectrophoretic microfluidic system imitates the oviduct of mammals with a microchannel to achieve fertilization in vitro (IVF) of an imprinting control-region (ICR) mouse. We applied a microfluidic chip and a positive dielectrophoretic (p-DEP) force to capture and to screen the sperm for the purpose of manipulating the oocyte. The p-DEP responses of the oocyte and sperm were exhibited under applied bias conditions (waveform AC 10 Vpp, 1 MHz) for trapping 1 min. The insemination concentration of sperm nearby the oocyte was increased to enhance the probability of natural fertilization through the p-DEP force trapping. A simulation tool (CFDRC-ACE+) was used to simulate and to analyze the distribution of the electric field. The DEP microfluidic devices were fabricated using poly (dimethylsiloxane) (PDMS) and ITO (indium tin oxide)-glass with electrodes. We discuss the requirement of sperm in a DEP microfluidic chip at varied concentrations to enhance the future rate of fertilization in vitro for an oligozoospermia patient. The result indicates that the rate of fertility in our device is 17.2 ± 7.5% (n = 30) at about 3000 sperms, compatible with traditional droplet-based IVF, which is 14.2 ± 7.5% (n = 28).

Pre-Implantation Mouse Embryos Cultured In Vitro under Different Oxygen Concentrations Show Altered Ultrastructures

Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O2) concentrations to culture human embryos. Overall, embryos cultured under physiological O2 tension (5%) have improved development compared to embryos cultured under atmospheric O2 conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O2 (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O2 tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O2 results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O2 showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O2 tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O2 is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O2.

Comparison of letrozole with gonadotropin-releasing hormone agonist in frozen embryo transfer after recurrent implantation failure: A randomized control trial

Background: The use of frozen embryo transfer (FET) is increasing worldwide in the treatment of infertility by in vitro fertilization. Different methods of endometrial preparation for FET have been suggested. Objective: The aim of this study was to compare the pregnancy outcomes after treatment with letrozole and those after treatment with the combination of gonadotropin-releasing hormone (GnRH) agonist and estradiol in FET. Materials and Methods: This randomized controlled trial study was conducted on 142 infertile women with a history of previous FET failure. Subjects were randomly assigned to one of two groups (n = 71 each). The GnRH group received 500 µg of buserelin plus 4mg estradiol (which increased to 8 mg if endometrial thickness was less than 5 mm), and the letrozole group received 5 mg of letrozole plus 75 IU of recombinant human follicle-stimulating hormone). At least two high-quality embryos were transferred to each subject in both groups. The outcome measures were clinical pregnancy rate and fetal heart rate detection Results: Subjects in the study groups had similar demographic characteristics and baseline clinical condition. Mean endometrial thickness in the letrozole and GnRH agonist groups were 8.90 ± 0.88 mm and 8.99 ± 0.85 mm, respectively (p = 0.57). The number of positive results of the beta human chorionic gonadotropin test and detection of fetal heartbeat were not significantly different between the groups (p > 0.05). Conclusions: The administration of letrozole and GnRH may produce similar pregnancy outcomes in FET. Key words: Letrozole; Fertilization in vitro; Pregnancy outcome.