Roger A Pierson
Department of Obstetrics, Gynecology and Reproductive Sciences, College of Medicine, University of Saskatchewan, Royal University Hospital, Saskatoon, Saskatchewan, Canada Imaging has become such an integral part of clinical care in the assisted reproductive technologies that it is difficult to imagine how in vitro fertilization IVF was done before we had the ability to visualize the ovaries and uterus easily. Recall that IVF was once done using laparoscopic retrieval of oocytes following...
Motion Analysis
Motion analysis, or direct measurement of subendometrial contractions, is a method of evaluating the endometrium based on the observation that the Figure 5 Midsagittal view of a uterus with a pronounced intraluminal fluid collection. The cervix is visualized to the left of the image and the fundus to the right. Fluid collections on the day of embryo transfer are associated with a very low probability of pregnancy. uterus and endometrium are in constant motion 110-117 . Objective assessment of...
Buffer System
Most embryo media utilize a bicarbonate CO2 buffer system to maintain a physiological pH of between 7.2 and 7.4 in the medium. The inclusion of sodium bicarbonate in the medium requires the use of a CO2 incubator to maintain a 5-7 CO2 atmosphere. An advantage of the bicarbonate CO2 system is that it is the physiological buffering system in fluid surrounding mammalian cells. A major drawback, however, is the rapid pH increase that occurs when medium is exposed to air, resulting in impaired...
Embryo Metabolism
Despite the fact that numerous morphological criteria have been published which could add to predictive power on further developmental potential of day 3 embryos, there is a tendency to question a close correlation between overall day 3 morphology and blastocyst formation as well as quality 101-103 . In this regard, biochemical criteria could be more appropriate to filter out those embryos with metabolic activity within normal range, and thus identify those embryos which will preferentially...
NORMAL VS ABNORMAL FERTILIZATION Cell Signaling Events
In humans, as all other mammalian species studied so far, normal fertilization is characterized by a particular type of calcium signals consisting of calcium oscillations and calcium waves refer to previously discussed section Sperm-Oocyte Fusion and Oocyte Activation . The same form of calcium signals was observed after conventional IVF 4 , subzonal insemination 18 , ICSI 5,18 , and round spermatid injection 19 . Animal experiments have suggested that the quality of the signal encoded by the...
Sperm Penetration Through Oocyte Vestments
Before the fertilizing spermatozoon reaches the oocyte, it has to penetrate two vestments, the cumulus oophorus and the zona pellucida. Sperm passage through the oocyte vestments involves receptor-mediated interactions with molecules present in the vestments, mainly progesterone in the cumulus oophorus and the ZP3 glycoprotein in the zona pellucida reviewed in Ref. 1 . These interactions are needed to prepare the spermatozoon for the final phase of fertilization this preparation is commonly...
Follicle Development and Oocyte Maturation In Vivo
The development of human oocytes is arrested at the prophase I stage of meiosis during fetal life. At birth, there are approximately one million primordial follicles in the ovaries 9 , each of which consists of an oocyte surrounded by a few flattened pregranulosa cells enclosed by a basement membrane. Although large numbers of follicles can leave the primordial pool and begin to grow, very few will be selected to mature and to ovulate for potential fertilization. Follicles respond to rising...
IVM and Preimplantation Genetic Diagnosis
Preimplantation genetic diagnosis PGD is a procedure whereby embryos produced by couples who are at risk of having children with an inherited disease or genetic defect, or by patients who have had three or more unexplained miscarriages, can be tested prior to implantation. Couples can therefore choose to have only those embryos diagnosed as being unaffected implanted in the woman's uterus, thus improving the chances of a successful pregnancy. IVF is normally necessary for patients who elect to...
ICSI Procedure
For the ICSI procedure itself, an inverted microscope equipped with micromanipulators and microinjectors should be available 63 . Magnification capability of 200 x and 400 x is a prerequisite for precise procedures such as ICSI. A heating stage on the inverted microscope maintains the temperature at 37 C. Ambient temperature control is of vital importance for the survival of oocytes, which are very sensitive to a decrease in temperature that can cause irreversible damage to the meiotic spindle...
Incubation VolumeEmbryo Grouping
Within the lumen of the female reproductive tract, the developing embryo is exposed to microliter volumes of fluid 191 . In contrast, the embryo grown in vitro is subject to relatively large volumes of medium up to 1 mL. Consequently, any autocrine factor s produced by the developing embryo will be diluted and may therefore become ineffectual. It has been demonstrated in the mouse that cleavage rate and blastocyst formation increase when embryos are grown in groups up to 10 or reduced volumes...
Introduction Sna
Human spermatozoa at ejaculation are incapable of in vivo fertilization and must undergo maturational change during which they acquire the ability to fertilize oocytes. This process, known as capacitation, was described more than 50 year ago by both Austin 1 and Chang 2 . Capacitation is prevented in ejaculated spermatozoa by at least one factor in seminal plasma 3 . Additionally, prolonged exposure to seminal plasma can inhibit the ability of spermatozoa to undergo the acrosome reaction in...
Normal Fertilization
However, the vast majority of zygotes will present with two centrally aligned pronuclei 18 to 20 hours post-insemination. Within these pronuclei, nucleoli tend to align at the pronuclear junction, but since this condition is time-dependent 51 , embryologists may be confronted with various pronuclear patterns at the time of fertilization check. Scott and Smith 45 were the first to report a prognostic value of a zygote score involving pronuclear appearance on implantation and delivery rate. This...
Immature Oocyte Retrieval
Oocyte retrieval is done under spinal anesthesia or intravenous sedation using fentanyl and midazolam 1-2 mg . Intravenous fentanyl is administered at intervals of 15-20 min up to a total dose of 150-200 mg. Local infiltration of bupivacaine 0.5 in the vagina reduces the discomfort of multiple needle punctures. Retrieval is performed under ultrasound guidance with a 19-G, single-lumen aspiration needle. The aspiration pressure is reduced to 7.5 kPa. The follicular fluid is collected in culture...
Ivm Treatment Outcome
Pregnancy rates with IVM are correlated with the number of immature oocytes retrieved. In women younger than 35 years from whom we retrieved more than 10 immature GV oocytes, we have achieved a clinical pregnancy rate of 38 per cycle. With an oocyte retrieval rate of more than 50 from the follicles present, women with 20 or more follicles at the baseline scan for IVM would be the best candidates for IVM. Our implantation rates are approximately 12 . As with IVF, clinical pregnancy and...
Modification of the ICSI Procedure
There is a body of evidence indicating that complete absence of pronuclear formation after ICSI is mostly caused by a failure of oocyte activation 55,56 reviewed in Ref. 11 . Globozoospermia round-headed sperm syndrome is a well-defined condition in which the ability of spermatozoa to activate the oocyte is known to be absent or severely impaired 52 , although spermatozoa from some globozoospermic patients fertilize normally 57 . Moreover, cases of sperm-oocyte-activating factor deficiency,...
Fertilization and Embryo Cleavage After ICSI
After the injection procedure, oocytes are rinsed and cultured in micro-droplets covered with lightweight paraffin oil. The conditions are similar to those employed for IVF inseminated oocytes the oocytes are kept at 37 C in an atmosphere of 5 O2, 5 CO2, and 90 N2. Injected oocytes are examined for integrity and fertilization about 16-18 hours after ICSI 67 . An average damage rate of approximately 9 of the injected oocytes can be expected, irrespective of the origin of the sperm used 45 ....
Abnormal Findings
At least in terms of oocyte polarity, a good quality two-pronuclear zygote is characterized by two polar bodies being located near the pronuclear axis 42 . Any deviation from this presumed optimal arrangement that cannot be corrected by microtubuli-driven rotation of the pronuclei 43 could lead to embryos of reduced morphology 42 . This drawback is in line with a high rate of complex genetic abnormalities found in embryos derived from zygotes with impaired polarity 44 . However, it may happen...
SpermOocyte Fusion and Oocyte Activation
After sperm penetration through the zona pellucida, the sperm plasma membrane fuses with the oocyte plasma membrane in a narrow equatorial region of the head, followed by incorporation of the whole spermatozoon to the oocyte by a phagocytosis-like process 1 . All components of the spermatozoon, with the exception of nuclear DNA, are subsequently dismantled in the oocyte cytoplasm. An immediate consequence of sperm-oocyte fusion is oocyte activation 2 . Shortly after the fusion, a particular...
Anesthesia or Analgesia
Already from the beginning our group performed TVOR combined with conscious sedation. Our reason was driven by the belief that if TVOR could be accepted by the patients and performed under local anesthesia, it would bring down the cost of the whole IVF treatment. Oocyte retrieval is supposed to be physically the most painful part of the IVF procedure, and various methods of analgesia have been tried over the years. Many groups therefore use general anesthesia. In our opinion, one of the leading...
Density Gradient Centrifugation
Density gradients may be either continuous or discontinuous although the discontinuous gradients have been used almost exclusively since the late 1980s 21 . Discontinuous gradients are usually prepared with two or three layers. Colloidal silica with covalently bound silane molecules is probably the most common density gradient material currently used for clinical IVF and andrology. PureSperm NidaCOn International AB, Goteborg, Sweden , Isolate Irvine Scientific, Santa Ana, California, U.S.A. ,...
References
1. Vermesh M, Kletzky OA. Longitudinal evaluation of the luteal phase and its transition into the follicular phase. J Clin Endocrinol Metab 1987 65 653. 2. Gorgeon A, Lefevre B. Evaluation of the largest healthy and atretic follicles during the human menstrual cycle. J Reprod Fertil 1983 69 497. 3. Pache TD, Wladimoroff JW, de Jong FH, Hop WC, Fauser BCJM. Growth patterns of non-dominant ovarian follicles during the normal menstrual cycle. Fertil Steril 1990 54 638. 4. Tsafriri A, Adashi EY....
Fixed Versus Flexible Administration of the Antagonist
In the first study using GnRH-nt, the protocol included a fixed day of GnRH-nt administration. In the multiple-dose protocol, the antagonist Figure 3 Gonadotropin-releasing hormone antagonist multiple- and single-dose protocols. Fixed day regimens. Abbreviations FSH, follicle-stimulating hormone hCG, human chorionic gonadotropin hMG, human menopausal gonadotropin. Figure 3 Gonadotropin-releasing hormone antagonist multiple- and single-dose protocols. Fixed day regimens. Abbreviations FSH,...
References Kkh
1. Yanagimachi R. Mammalian fertilization. In Knobil E, Neill JD, eds. The Physiology of Reproduction, 2nd ed. New York Raven Press, 1994 189-317. 2. Jones KT. Ca2 oscillations in the activation of the egg and development of the embryo in mammals. Int J Dev Biol 1998 42 1-10. 3. Cuthbertson KS, Cobbold PH. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2 . Nature 1985 316 541-542. 4. Taylor CT, Lawrence YM, Kingsland CR, Biljan MM, Cuthbertson KS....
SPERM COLLECTION Ejaculation
The semen specimen should be collected by masturbation and the ejaculate produced into a sterile glass or disposable plastic jar that has been checked for sperm toxicity. As soon as the seminal plasma has liquefied, the specimen should be analyzed according to the WHO guidelines 6 and prepared for sperm isolation. A second semen specimen may be requested if the semen specimen on the day of IVF is of very poor quality 7 . When liquefaction is delayed or the specimen is especially viscous,...
Hormones and Growth Factors
Although the mouse blastocyst is capable of metabolizing exogenous steroid hormones 225 , there is limited data on the direct action of hormones on the early embryo 226-229 . Certainly, estradiol appears to have a direct negative impact on mouse embryo development 230,231 . However, prolactin at a concentration of 300 ng mL, has been shown to improve the rate of blastocyst formation from cultured 2-cell mouse embryos 232 . Available evidence indicates that the effects of maternal hormones on...
Spectral Doppler and Color Flow Doppler Ultrasonography
The history of Doppler ultrasonography of the uterine arteries in the literature is confusing because many reports failed to differentiate between spectral Doppler and color flow Doppler imaging. Early studies tend to be based on spectral Doppler examinations, which are a means of evaluating the resistance to blood flow using calculations of the pulsatility index PI , resistance index RI , Vmax, or the systolic-to-diastolic ratio S D ratio . Color flow Doppler and power flow Doppler imaging are...
References 1
1. Karten MJ, Hoeger CA, Hooh WA. The development of safer antagonists strategy and status. In Bouchard P, Haour F, Franchimont P, Schatz B, eds. Recent Progress on LH-RH and Gonadal Peplides. Paris Elsevier, 1990 147-158. 2. Reissmann T, Felberbaum R, Diedrich K, Engel J, Comaru-Schally AM, Schally AV. Development and applications of luteinizing hormone-releasing antagonists in the treatment of infertility an overview. Hum Reprod 1995 10 1974-1981. 3. Matikainen T, Ding YQ, Vergara M,...
GnRH AgonistFSHShort Boost Protocol
This protocol Fig. 3 is typically reserved for poor responders, namely patients with day 3 FSH gt 10 IU L and patients with a previous IVF cycle in which they have been cancelled or less than three oocytes were retrieved poor responders'' or women who previously had a live delivery with a boost cycle. This protocol boosts the exogenous gonadotropin injected Figure 3 A diagram of the procedures, monitoring, and medicines used for short or Boost protocol commencing on day 2 of the menstrual...
Assessment of Ovarian Follicular Reserve
Changes in demographic trends in the age at first pregnancy in our times have combined to yield more and more women seeking pregnancy when they are older and less fertile. Numerous studies in recent years have demonstrated that fertility declines progressively as age advances. In IVF, the main focus of attention is on assessment of what is termed the ovarian reserve. Ultraso-nography is now being used to investigate follicular dynamics in aging women as are detailed endocrine-based tests 24 . A...
QUALITY CONTROL Mouse Bioassay
The preimplantation mouse embryo is the most widely used bioassay for medium components, culture media, and equipment used in clinical IVF. Using mice for testing media for human embryos has been the focus of much discussion due to conflicting reports in the literature of its suitability as a bioassay 310,311 . Fukuda et al. 312 reported that for the mouse, in vitro fertilization and the development of zygotes and 2-cell embryos in culture was positively correlated with the purity of the water...
Poor Responders
Poor responders to exogenous gonadotropins require markedly increased quantities of gonadotropins to obtain multifollicular development. Moreover, the number of oocytes, fertilization rates, embryo quality, and pregnancy rates are all decreased in this patient population. The incidence of poor response varies from 5 to 18 of unselected IVF patients 70 . Poor response to gonadotropins is a result of diminished ovarian reserve. The most important cause is advanced female age. An age-related...
Perifollicular Blood Flow Doppler Indices
1. Serhal PF, Ranieri DM, Kinis A, et al. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod 1997 12 1267-1270. 2. Ebner T, Moser M, Sommergruber M, et al. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development. Hum Reprod Update 2003 9 251-262. 3. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod 1996 11 1975-1978. 4. Van Blerkom J,...
Ovarian Hyperstimulation Syndrome
OHSS is the major serious and potentially life-threatening complication of ovulation induction in IVF-ET treatment. It is characterized by transudation of protein-rich fluid from the vascular space into the peritoneal cavity and to a less extent, pleural and pericardial cavities. The basic pathophysio-logic event in OHSS is an acute increase in capillary permeability however, the exact factors responsible for this phenomenon have, until recently, not been clear. Because intensity of the OHSS is...
The Blastocyst
After compaction of the cleaving embryo, it begins to form a cavity. During blastocyst formation, two clearly distinguishable cell lines are formed, namely the trophectoderm and the inner cell mass. In humans, the proliferation of the latter was found to be 1.5 times lower than that of the trophectoderm 107 . Similar to the developmental stage of the blastocyst on days 5 and 6, which may range from a retarded morula to an expanded or hatching blastocyst, a high variability in cell number has...
Antioxidants
It has been proposed that one of the causes for the retarded development of preimplantation embryos in culture compared to those developed in vivo is oxidative stress. Potential sources of such stress include the use of high oxygen tension i.e., 20 , exposure to light, and the presence of transitional metals in the culture medium 162 . Therefore, several studies have examined the effect of known antioxidants on preimplantation embryo development, although the data to date remain rather...
In Vitro Fertilization The First Three Decades
International Federation of Fertility Societies, European Society of Human Reproduction and Embryology, Paris, France Eastern Virginia Medical School, Norfolk, Virginia and Johns Hopkins University Hospital, Baltimore, Maryland, U.S.A. The birth of the world's first baby born as a result of in vitro fertilization IVF in July 1978 was by no means a chance event. Indeed, in the long evolution of reproduction, conception by IVF represents the end of a continuum which originated with childbirth...
Embryo Culture Protocol
The protocol described below is based on the assumption that the media in question will be renewed after 48 hours. As discussed above, this is paramount whether one is using a biphasic or non-sequential system. It has been validated using the media and consumables listed. Any change to the protocol, whether it be a different source of oil or media needs to be validated carefully. When using the sequential media G1 and G2 131 , embryos should be cultured in a gas phase of 5 O2 and 6 CO2....
Sperm Migration
Motile spermatozoa separate themselves from seminal plasma in vivo by actively migrating through cervical mucus in the female reproductive tract. There are a variety of sperm preparation techniques that involve migration of spermatozoa, and the element common and prerequisite to all these techniques is the self-propelled movement of spermatozoa. The swim-up of spermatozoa from a washed pellet technique was originally described by Mahadevan and Baker 11 and it is still a standard method for...
ImagingBased Uterine Scoring System
An imaging-based scoring system to predict uterine sensitivity has been proposed which appears to be based on an earlier uterine biophysical profile system 103,104 . The scoring system was designed by assigning points for various criteria and then adding the cumulative columns. Comparisons of uterine scores in conception with non-conception cycles demonstrated no differences in any criteria measured, including endometrial thickness, endometrial pattern, PI, RI, color Doppler, and other vascular...
Cycle Monitoring
NICE guidelines recommend ultrasound monitoring only to measure follicle size and follicle number for patient management during multi follicular folliculogenesis for IVF. Specifically, serum E2 monitoring is not recommended. We have minimized the use of serum E2, P4, and LH measurements during cycle monitoring in our IVF program and still find these helpful in our IVF program for basic ovarian stimulation. We do accept that experienced IVF units may have no difficulty in preventing ovarian...
Types of Media for Embryo Culture
Culture media employed for clinical IVF vary greatly in their composition, yet there appears to be little difference between media in their ability to support development of the human embryo in vitro for up to 48 hours or in subsequent pregnancy rates after transfer 12 . This has led to a great deal of confusion concerning the formulation of embryo culture media and the role of individual components in embryo development. An understanding of the role of culture media and their components has...
Testing Ovarian Reserve
Atlanta Center for Reproductive Medicine, Atlanta, Georgia, U.S.A. Both the quantity of eggs and their quality are strong influences on IVF outcome. Markers of ovarian reserve, such as basal follicle-stimulating hormone FSH and basal antral follicle BAF counts, are good predictors of the quantity of eggs which can be induced to grow. However, the quality of those eggs seems better predicted by the age of the women. In women past age 40, current success rates are low overall, even among those...
Endometrial Preparation and Luteal Support
To achieve optimum endometrial growth, exogenous estradiol 17 p micro-nized is started on the day of retrieval. The dosage is determined by endometrial thickness measured on the day of retrieval. If the endometrial thickness is less than 6 mm, then 12 mg a day is started if the thickness is between 6 and 8 mm, then 10 mg a day is started and if the thickness is more than 8 mm, then 6 mg is used, all in three divided doses. Recently, we have begun administering the estradiol treatment even...
Zygote Morphology
Formation and differentiation of pronuclei are the most prominent morphological events taking place in the early postfertilization period. This process was studied in detail by electron microscopic analysis of male pronuclear development in polyspermically penetrated human zona-free oocytes 9 . It was found that the assembly of NPBs in the pronuclei was dependent on RNA synthesis 29 , and on the activity of ooplasmic factors 30 . Cortical granule exocytosis and redistribution of cytoplasmic...
Endometrial Thickness and Pattern for Assessing Endometrial Receptivity
Ultrasonography has been used, with varying degrees of success, to correlate the probability of pregnancy in ovarian stimulation-ovulation induction cycles and IVF cycles 73-77 . Most imaging studies have been attempting to predict the probability of implantation. A thicker endometrium was observed on the day of oocyte retrieval in women who conceived during that cycle 74 . The IVF pregnancy rate increased in cycles when the endo-metrium was gt 9 mm but lt 14 mm 75 . In another study, no...
Indications for ICSI
Before the era of ICSI, attempts were made to modify and refine conventional IVF to achieve increased rates of conception in cases of male-factor infertility. Today, ICSI has clearly overshadowed the use of modified IVF procedures including high insemination concentration for the treatment of severe male-factor infertility. ICSI requires only one spermatozoon with a functional genome and centrosome for the fertilization of each oocyte. Indications for ICSI are not restricted to impaired...
Contributors
Hanadi Ba-Akdah Department of Obstetrics and Gynecology, McGill University, Montreal, Canada Gordon Baker Melbourne IVF, East Melbourne, Victoria, Australia Nancy L. Bossert Reproductive Medicine Center, University of Minnesota, Minneapolis, Minnesota, U.S.A. Sue Breheny Monash IVF, Epworth Hospital, Richmond, Victoria, Australia Ri-Cheng Chian Department of Obstetrics and Gynecology, McGill University, Montreal, Canada Jacques Cohen Tyho-Galileo Research Laboratories and Reprogenetics, West...
Evaluation and Preparation of the Infertile Couple for In Vitro Fertilization
Reproductive Partners Medical Group, Redondo Beach, California, U.S.A. Thorough evaluation of the infertile couple before in vitro fertilization IVF is critical in achieving the best outcomes and avoiding complications. Most IVF centers organize the evaluation by using a checklist that the nurse coordinator and physician assure is complete before proceeding with the cycle. A level exceeding 25mIU ml about 12mIU ml using current assays has been correlated with a very low chance of pregnancy 1 ....
IVM for Poor Responders
Poor response to gonadotropin stimulation occurs more often in older women but may also be present in young women, including those with normal endocrine profiles as well as those with abnormal endocrine parameters namely, high baseline FSH and estradiol E2 levels known to be associated with poor response. Some poor responders appear to respond to stimulation but have a low estrogen level, whereas others have few or slow-growing follicles. Normally, these patients require prolonged stimulation...
Complications
In this part, I will only deal with complications related to TVOR because this is the gold standard today for oocyte collection in ART. Complications related to laparoscopic procedures are dealt with elsewhere. Despite all the advantages with TVOR during IVF treatment, the aspiration needle may injure pelvic organs and structures leading to serious complications. The most common complications are hemorrhage, trauma, and injury of pelvic structures and pelvic infection. Other complications...