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# Title
1 What is IVF?
IVF is an acronym for in vitro fertilization ('in vitro' meaning 'in glass'). Simply put IVF is adding a man's sperm to his female partners eggs in the laboratory to produce embryos. In vitro fertilization is an option for many couples who cannot conceive through conventional therapies. These embryos are put back into the female partner's uterus (womb) after 3 to 5 days of being in the incubator, hopefully they will then grow into a baby. The reasons IVF is done include - poor sperm quality and/or quantity, obstructions between the egg and sperm, ovulation problems, and sperm-egg interaction problems. These problems can prevent couples having a baby naturally, and IVF helps to solve this. Specific conditions that might require IVF include: Tubal blockage or failed tubal reversal Endometriosis Cervical factor Pelvic adhesions Male factor Unexplained infertility/ failed conventional therapy Genetic testing (PGD) for inheritable diseases Genetic testing (PGD) for possible reasons for multiple miscarriages The first IVF baby was Louise Brown, born at 11:47 p.m. on July 25, 1978 at Oldham General Hospital, Oldham, England through a planned caesarean section. She weighed 5 pounds, 12 ounces (2.608 kg) at birth. Dr. Patrick Steptoe, a gynecologist at Oldham General Hospital, and Dr. Robert Edwards, a physiologist at Cambridge University, had been actively working on finding an alternative solution for conception since 1966.VideoThe video shows a conventional insemination with the egg surrounded by coronal and cumulus cells and sperm swimming around the egg. The video shifts focus over time from the egg to the sperm.
2 LifeIn Interview : Interviewing Experts of ART: An unique and Innovative Initiative
A noble initiative by Lifeinvitro team – LifeIn Interview – Experts in reproductive medicine/science answer the day to day problems in infertility and assisted conception for a better management of infertile couples.  Website Link is: www.lifeinvitro.com/p/interviews.shtml This month’s interview is with Prof. Kathryn C. Worrilow (Ph.D).    Questions have been asked:    1. Provide an overview of Cleanroom classifications. 2. How is filtered and pressurized air generated for an IVF laboratory? 3. Is a HEPA filter sufficient for cleanliness or is a VOC filter necessary also? 4. How can VOCs affect the embryo development? 5. Do all labs have a dedicated HVAC system? Some labs even keep air conditioners inside the IVF laboratory!! Is it true that a window AC unit pushes contaminated outdoor air into the lab as cold air? 6. Give an overview of the different classes of Laminar Air Flow (LAF) hoods. LAF hoods are the main work area in most IVF labs. However, it has been noted that some people keep the airflow off always. Is it right approach? 7. What are the pros and cons of using UV light irradiation in the lab? Some of the labs’ air handling unit outside the lab has a HEPA filter and UV light. Is there a particular “smell” in the lab if the UV light is kept on? Does UV light cause any toxic reactions in the air? 8. Nowadays different levels of air purifiers are available for labs. Stand alone systems (with VOC filter) to be kept inside the lab, inline filters for gas lines, VOC filter unit for incubators? Which are the ideal options? 9. How to measure air contaminants in the beginning of establishment of unit and later on regular intervals? 10. What is the best way to control air contaminants in the IVF lab area? 11. How to control microbial contaminants in the lab area? And how to monitor them? This is a major issue in tropical climates. 12. Can fungal growth happen in the HEPA units of HVAC system or LAF? 13. On what intervals should one change the filters? 14. Advice to lab personnel!! Hope the professionals would be befitted. This is not a place for any kind of advertisements. Medical Information Disclaimer: "This Interview has been done through online and the responses are completely based on interviewee's best current knowledge. Lifeinvitro is broadcasting all the content of the interview as it is. Lifeinvitro does not have any liability on the authenticity of the information. This is completely an expert's view. This information is intended as a professional education resource only and should not be used for self diagnosing or self treating a health problem as it is not a substitute for expert professional care. If you find this interview contains erroneous/outdated information or if you have any suggestion, please write to us at admin@lifeinvitro.com stating in subject line ‘Letter: Lifein Interview’.”
3 Embryo Development
Oocyte (Egg) stages Germinal Vesicle (Immature) The germinal vesicle, indicated by the arrow, shows this egg to be in prophase I of the first meiotic division. (Please note that eggs and embryos are usually surrounded by a layer of corona cells, these look like a halo around the egg when spread out. The cumulus cells spread out even further and generally have the appearance of a fluffy cloud. The cumulus can often be seen without a microscope. The egg together with these cells is sometimes called the oocyte-cumulus-corona cell complex. In many of the pictures below the cumulus and coronal cells have been removed leaving the bare egg/embryo. The cumulus and coronal cells are either removed prior to ICSI so maturity can be assessed and so a good seal can be achieved between the egg and the ICSI holding pipette. The enzyme used to remove the cumulus and coronal cells is Hyaluronidase. In the case of conventional insemination - sperm added to the media and allowed to penetrate the egg naturally - the cumulus and coronal cells are digested by the sperm and the remaining cells are removed when checking for fertilisation)Metaphase 1 (Immature) The germinal vesicle is no longer visable and a polar body has not yet been extruded. Metaphase 2 (Mature) In these pictures we can clearly see a polar body as indicated by the arrow. The polar body is a sign that the egg has reached metaphase 2 in it's development, and that the egg is mature and ready to fertilise with either conventional insemination techniques (adding sperm to the media surrounding the egg) or ICSI (injecting a sperm directly into the center of the egg). Embryo Stages 2PN Stage (Normal Fertilization - day 1 after insemination) Below we can see a normally fertilised egg. The arrow indicates the two circles (pronuclei) containing small dots (nucleoli). One of the pronuclei contains genetic information from the egg the other from the sperm. It is thought that polarity of the pronuclei and even distribution of the nucleoli between the two pronuclei indicates the potential quality of the developing embryo. The pronuclei are usually checked between 16-18 hours after the sperm is added to the egg. The 2PN will eventually disappear with the union of two gametes this is known as Syngamy. The zygote is now on it's way to become an embryo. Day 2 after insemination (2 - 4 Cell Embryo) 24 hours after the 2PN were seen the embryo has already started to divide. Blastomeres often divide evenly,although asynchonously, so it is not uncommon for embryos to have an odd number of cells and uneven blastomeres. We can also see various indicators of poorer quality in the embryo such as fragments or multinucliation of blastomeres. Day 3 after insemination (8 Cell Embryo) Approximately 72 hours after the eggs were first inseminated they will usually have reached around the 8 cell stage. In the IVF program it is common to transfer the best of the embryos from a treatment cycle back to the uterus at this stage. If the patient will be having preimplantation diagnosis to test the genetic makeup of the embryo, the biopsy (removing a cell from the embryo to test) will usually be performed on this day. Day 4 after insemination (Morula) The embryo continues to divide, and there is increased cellular adhesion until the distiction between the blastomeres becomes difficult to see. The embryo at this stage is called a Morula. Day 5 after insemination (Blastocyst) The morula will continue to develop and start to cavitate. The embryo begins to turn into a hollow ball of cells known as a blastocyst. The blastocyst consists of two main parts an area denser with cells called the inner cell mass (ICM) which will eventually form the fetus, and the trophectoderm which will go on to be the placenta. The picture shows two blastocysts, the one on the left is an expanded blastocyst, the one on the right has expanded and is hatching from the shell (zona pellucida) and is getting ready to implant.
4 IVF Ethics
IVF has always been controversial, Patrick Steptoe and Robert Edwards were faced with scores of people claiming things like ' they were playing God' and that 'any babies produced would not have a soul'. Fortunately when Louise Brown was born and people saw a healthy baby, many of these issues were forgotten. However, as IVF technology develops more and more ethical questions are raised. IVF is now clearly accepted by the majority of the population and looking back on recent IVF history it does seem that ethical considerations and opinions are changing. Just a few years ago treatment for single women and lesbian couples was very difficult to find, as was sex selection, now it is easily possible to find clinics who will accomodate these wishes. The following questions are not answered (occasionally points of interest are added in italic blue). Comments are invited below. IVF Issues At what point should pre-embryos/embryos be considered to have human rights? Is "creating", discarding, freezing, or manipulating them right? Pope Benedict XVI stated in June 2006, " The human being has the right to be generated, not produced, to come to life not in virtue of an artificial process but of a human act in the full sense of the term: the union between a man and a woman". " Never before in history has human procreation, and therefore the family, which is its natural place, been so threatened as in today's culture. Procreation must always take place within the family." " They need to be aware that true love is only that which comes from the union of a man and a woman." " A true family comes from the union of two people from different sexes." Who should decide what is appropriate in IVF? Politicians? The Church? Scientists? Should single women be allowed access to IVF in order to have children? Is there a need for a father? Should homosexual male or female couples be allowed access to IVF treatment in order to have children? UK MPs (June 2006) have called for an end to the right of fertility clinics to refuse treatment to single women and lesbians. The debate was triggered by the Commons science and technology committee report in 2005 on the issue, who described the current rules regarding unconventional families as "offensive". Is it responsible to allow so many multiple pregnancies or reductions due to IVF treatment? UK law now tries to limit this and only permits a maximum of 2 embryos to be transferred except in very special circumstances. Who should be responsible for funding IVF? Patients? Insurance companies? Healthcare systems? How long should embryos be allowed to be frozen? If they go past that time should they be destroyed? Should frozen embryos be destroyed if patients stop paying the storage fees?   Preimplantation Genetic Diagnosis (PGD). PGD is used to test embryos prior to transfer into the uterus. Part of the embryo is used for this procedure and is removed from the embryo. This selection of "healthy" embryos is sometimes referred to in news articles as "Designer Embryos". So should PGD be used at all? Should PGD be used only for detecting very serious, life threatening conditions and not for minor genetic abnormalities? This is the current advise of the Human Fertilisation and Embryo Authority, UK. Is it OK to genetically select an embryo which would be a near perfect match for a sibling with a life threatening disorder? Should PGD be only available to those who can afford it? Is sex selection wrong? Is selecting embryos with certain traits or characteristics wrong?   Cloning and Stem cells Is there ever a case where human reproductive cloning is acceptable? The production of a new life? Replacing a dead child? Is it OK to use cloning technology to create stem cells (therapeutic cloning) to help cure disease? Should cloning research be regulated? Who should police it? Can this be achieved on a global basis? If potential parents can only have a child through cloning do they have that right? What are the physical and psychosocial consequences of cloning on the child? What is the impact on familial and societal relations? What will be the potential effects on the human gene pool?
5 Embryo Freezing & Thawing
Embryo Thawing Cryopreservation of embryos has always been an important tool in an IVF program. It enables a precautious policy for embryo transfer, lessening the chance of multiple pregnancies, knowing that the embryos not transferred will be available for subsequent thawed cycles. This also establishes a cumulative pregnancy rate, increasing the overall chances for patients to conceive per IVF cycle, as well as helping patient management with complications such as Ovarian Hyperstimulation Syndrome (OHSS). However, results from frozen/thawed cycles are often disappointing with success rates usually around half that of a fresh cycle. Embryos are placed in a series of solutions to draw some of the water out by osmosis and then add a cryoprotectant. The removal of water helps prevent the formation of damaging ice crystals and the cryoprotectant protects the embryos during the freezing process. The embryo freezing process takes approximately 3 hours. The temperature is slowly decreased to -36?C and then plunged into and stored in liquid nitrogen at -196?C for long-term storage. Embryo Thawing Embryo thawing is the reverse of the freezing process, and involves warming the embryos to room temperature to allow the transfer back into culture media at 37?C in an incubator. The embryos are then ready for transfer to the uterus. The embryos are thawed either the day before or on the day of the scheduled embryo transfer. Embryo thawing takes approximately 2 hours. Sometimes individual cells within the embryo are damaged by the freezing process. Embryos with some freeze damage can still go on to produce a healthy pregnancy however the more the embryo is damaged the less likely it will be for the embryo to develop. Sometimes all of the cells within the embryo are damaged. In this situation the embryo will not be transferred.
6 Lysed Cell Removal (LCR) after freezing - thawing.
The process of freezing and thawing can be fairly harsh on the embryos and often not all of the cells or embryos survive. Lysed Cell Removal is a new technique that has been shown to dramatically increase the implantation potential of embryos that have been damaged by thawing. The technique known as Lysed Cell Removal (LCR) is giving improved results. It works by making a small hole in the zona pellucida with acid or laser then removing the cells that were damaged by the freezing which are thought to either disrupt the development of the embryo or produce negative factors as they degenerate.VideoThe video shows a small hole being made in the zona (egg shell) with a laser on the right hand side of the egg, then pipette is placed into the hole and lysed cells are aspirated. The embryo is then turned showing all traces of lysed cells have been removed. Video courtesy of Reproductive Biology Associates, Atlanta, Georgia.
7 ICSI - Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization procedure in which a single sperm is injected directly into an egg; this procedure is most commonly used to overcome male infertility problems. The cumulus cells that surround the egg are removed using an enzyme called hyaluronidase, this historically comes from bovine testis but new technology has provided a pure recombinant product that is now available. A small amount of washed sperm is placed into thick viscous media containing poly vinyl pyrrolidone (PVP) in a dish. The PVP slows the sperm down so that they can be morphologically assessed. The most normal looking sperm are selected and then immobilized by squashing their tales with a glass injection needle. The sperm are sucked into the needle tail first ready to be injected. The egg is then placed under a microscope and moved using micromanipulation devices (micromanipulators, microinjectors and micropipettes). A holding pipette stablizes the mature oocyte then from the opposite site a thin, hollow glass needle is pierced into the inner part of the oocyte. it is loaded with a single sperm that will be expelled into the oocyte. The pictured oocyte has an extruded polar body at about 12 o'clock indicating its maturity. After the procedure, the oocyte will be placed into cell culture and checked on the following day for signs of fertilization. In natural fertilization sperm compete and when the first sperm enters the egg cell, the egg cell blocks the entry of any other sperm. Concern has been raised that in ICSI this sperm selection process is bypassed and the sperm is selected by the embryologist without undergoing any specific testing. Reasons for ICSI: Sperm completely absent from the ejaculate (azoospermia) Sperm present in low concentrations (oligospermia). Poor sperm motility (asthenospermia) Poor sperm morphology (teratospermia) Sperm retrieved by surgical techniques (for example TESA, TESE) Problems with sperm binding to and penetrating the egg Antisperm antibodies Previous failed or poor fertilization Unexplained infertility Frozen sperm limited in quantity If preimplantation genetic diagnosis (PGD) is being used to screen embryos for a specific genetic disorder Video The video shows an egg held in position with a glass holding pipette on the left. A very fine glass injection needle containing an immobilized sperm penetrates the egg from the right. The injection needle breaks through the membrane and a small amount of cyoplasm is aspirated to confirm membrane breakage. The sperm is the inserted into the center of the egg and the needle removed.
8 Embryo transfer
The embryos are usually selected on day 3 or day 5 after retrieval. The number of embryos chosen for transfer will depend on a number of factors such as the maternal age, quality of embryos and results of previous cycles. After the transfer is complete the catheter is checked to ensure that none all the embryos are left behind. Embryo transfer can cause mild cramping. After transfer, the woman may get dressed and leave after a brief recovery period. A pregnancy test will be done twelve to fourteen days after the transfer, regardless of the occurance of any uterine bleeding. The transfer of several embryos increases the probability of success. A multiple embryo transfer also increases the risk of a multiple pregnancy. Any multiple pregnancy carries an increased risk of miscarriage(s), premature labor and premature birth as well as an increased financial and emotional cost. Video The video shows an embryo transfer by ultrasound. The uterus is in the middle with the cervix on the right hand side. The catheter enters the vagina on the right and we can see it pass through the cervix and into the uterus. A small amount of media is released containing the embryos followed by an air bubble, this can just be seen as a white flash. The catheter is then removed and the transfer is complete.
9 The oocyte/egg retrieval
Oocyte retrieval is performed approximately 36 hours after the hCG injection. The ovaries are scanned transvaginally then each follicle is then punctured using a long, sharp needle which is secured by a metal guide on the outside of the ultrasound probe. The needle punctures the wall of the vagina then into the ovary. As each follicle is punctured the fluid inside is remove with gentle suction, this aspirate is then passed into the laboratory where an embryologist will examine it carefully under a microscope to try to find an egg. If an egg is not found the follicle will be flushed with warm media to try to rinse out the egg. The eggs are then placed in an incubator at 37?C. Oocyte retrieval is usually performed under a light general anesthesia or local anesthesia.

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