Techniques For Getting Pregnant

Pregnancy Miracle By Lisa

The Pregnancy Miracle book details a highly unique program for treating infertility using traditional Chinese medicine and holistic methods. The author, Lisa Olson, offers easy-to-follow techniques and remedies involving diet, herbs and acupressure to improve your overall health and well-being, strengthen the organs and systems vital to reproduction, heal specific conditions that may affect fertility and even support reproductive methods such as IVF and hormone therapy. The Pregnancy Miracle system combines five secrets of ancient oriental medicine that restore the bodys balance when it comes to fertility. The book supplies information on how to correct many of the problems that affect both men and women when they are trying to get pregnant. If you are just getting started with your fertility journey, I highly recommend this book to be your first step. Exceptionally rich in information and actionable advice, it will educate and empower you, ultimately increasing the effectiveness of any treatment. Continue reading...

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Of Intracytoplasmic Sperm Injection ICSI

Intracytoplasmic sperm injection (ICSI) has been developed as an assisted reproductive technique by which a sperm is mechanically injected into the cytoplasm of an oocyte. This technique was pioneered initially in sea urchin (Hiramoto 1962), then in mouse (Lin 1966) and hamster (Ue-hara and Yanagimachi 1976). Although ICSI has been used successfully in humans throughout the world since it was first introduced clinically in 1992 (Palermo et al. 1992), ICSI in mouse proved extremely difficult, and was unsuccessful until the introduction of the piezo-driven micromanipulator in 1995 (Kimura and Yanagimachi 1995). In theory, ICSI of a single sperm, including sperm DNA and other sperm factors (Wilding and Dale 1997), whether live or amotile, is sufficient to derive a viable mouse. In general, ICSI results in a relatively high percentage (> 50 ) of successfully fertilized embryos and liveborn offspring, all of which are likely to wean and become reproductively sound. ICSI has been used to...

Reasonable Cautions and Concerns Regarding ICSI

ICSI is a remarkably effective ART for male mice with infertility or sperm dysfunction dysmotility due to any cause. However, by injecting a single sperm directly into the ooplasm of oocytes, the ICSI technique bypasses all natural sperm selection processes (cumulus penetration, zona binding, acrosome reaction, zona pellucida penetration, oolemma fusion, etc.). Consequently, the delivery of genetic material into an oocyte during ICSI raises concerns of contamination with pathogens and subsequent infection of the embryo. Another major concern is that of the genetic integrity of the sperm DNA. It is recommended that, whenever possible, motile sperm with normal morphology be used for ICSI. If the sperm has apparently normal morphology and is motile, there is a good chance of successful fertilization and embryo development. If the sperm is of very poor morphology or is largely amotile, then selection of the best sperm for ICSI becomes highly subjective. Structurally abnormal sperm are...

Remaining Questions On GnRHnt Pregnancy Rates

There is a trend in most of the controlled studies using GnRH-nt (with both compounds and protocols) to find slightly lower pregnancy rates as compared to the GnRH-a long protocol. This led to the questioning of IVF-ET results of GnRH-nts. A meta-analysis concluded there were significantly lower pregnancy rates in GnRH-nt cycles as compared to GnRH agonists (48). However, the difference was very close to being nonsignificant (OR 0.79 95 CI 0.630.99). Adding one study, Ludwig et al. (49) did not find a significant difference in pregnancy rates for Cetrorelix. A recent meta-analysis presented by Daya did not find a difference in ongoing pregnancy rate (unpublished data). Care should be taken in drawing conclusions on these observations. Some population factors were not equivalent in the groups despite randomization. In addition, the learning curve, inherent to the use of new treatment schemes, could have influenced some of the studied outcome. The trend towards higher pregnancy rate...

In Vitro Maturation Of Oocytes In Infertility Treatments

The research into maturation of immature oocytes initiated by Pincus and Enzmann (8) and continued by Edwards et al. (23) was not incorporated as a treatment for human infertility until 1991. Cha et al. (24) reported that human follicular oocytes were harvested from unstimulated ovaries during gynecological surgery, matured in vitro, then fertilized, and five embryos were transferred to a woman with premature ovarian failure. The recipient subsequently delivered healthy triplet girls. Trounson et al. (25) further suggested that immature oocyte recovery could be developed as a new method for the treatment of women with infertility due to PCO because the oocytes of these patients retain their maturational and developmental competence. However, the initial reported IVM pregnancy rates were low. Our group demonstrated that priming with hCG 36 hours prior to immature oocyte collection significantly improved the maturation rate, and the pregnancy rate exceeded 30 (26,27). IVM was initially...

Fertility Preservation

Oocytes could be harvested from ovaries of cancer patients after controlled ovarian hyperstimulation. However, there are two major drawbacks associated with conventional IVF first, the time interval needed for IVF ranges from 2 to 6 weeks beginning with the patient's next menstrual period, which may sometimes be too long due to the natural course of the malignant disease without therapy. Second, ovarian hyperstimulation is associated with high estradiol levels which may not be safe in some cases of estrogen-sensitive breast cancer. Ovarian stimulation for oocyte collection could be totally avoided by collecting immature oocytes (51). We recently reported the retrieval of immature oocytes from unstimulated ovaries before gonado-toxic therapy for oocyte vitrification purposes (52). This resulted in the successful preservation of fertility with no delay in chemotherapy, no surgery, and no necessity for hormonal stimulation. Since that report, 26 cancer patients have undergone immature...

Semen Cryopreservation and Assisted Reproduction

Cryostorage of semen has become standard practice and should be offered to all men before undergoing potentially sterilizing therapy. Improvements in the techniques used to store semen (49) and advances in the field of assisted reproduction, such as intracyto-plasmic sperm injection (ICSI), have increased the chance of successful pregnancies using cryopreserved sperm. However, there are some limitations to this method of preserving fertility. First, it is not a feasible option for prepubertal patients. Furthermore, testicular function in adult men with malignant disease is often impaired before treatment (9) resulting in poor sperm quality or difficulty providing semen for storage. Oligospermia is found in a third to a half of patients with Hodgkin's disease, NHL, and testicular cancer before treatment and also occurs in men with leukemia and soft-tissue cancer (7a). Sperm motility is also impaired in these patients, and the process of freezing and thawing semen further reduces the...

Intracytoplasmic Sperm Injection

Under these conditions, however, the importance of another set of factors, some of which were not perceived in the conventional IVF era because of their association with other defects that impeded sperm entry to the oocyte, gains importance. Most of these factors are related to success of oocyte activation or failure and to the early development of the zygote (11).

Intracytoplasmic Sperm Injection History of ICSI

Extremely low sperm counts, impaired motility, and abnormal morphology represent the main causes of failed fertilization in conventional IVF. Today, ICSI is the ultimate option to treat these cases of severe male-factor infertility. One single viable spermatozoon, preferably of good morphology, is selected by the embryologist and injected in each oocyte available. ICSI is based on micromanipulation of oocytes and spermatozoa. Initially, partial zona dissection (PZD) was established to facilitate sperm penetration (4-7). The barrier to fertilization represented by the zona pellu-cida was disrupted mechanically so that the inseminated sperm cells obtained direct access to the perivitelline space of the oocyte. Subzonal insemination (SUZI) represented the next step in micromanipulation techniques (8-11). SUZI enabled the immediate delivery of several motile sperm cells into the perivitelline space by means of an injection pipette. ICSI is even more invasive because a single spermatozoon...

Gamete Handling Prior to ICSI

Germinal Vesicle

A successful ICSI program depends on ovarian stimulation, which is essentially similar to methods used for conventional IVF. Current ovarian stimulation regimens use a combination of gonadotropin-releasing hormone (GnRH) agonists or antagonists, human menopausal gonadotropin (hMG), or recombinant follicle-stimulating hormone (recFSH), and human cho-rionic gonadotropin (hCG), which allows the retrieval of a high number of cumulus-oocyte complexes (43,44). Administration of GnRH agonists Fertilization by means of micromanipulation requires denudation of oocytes (i.e., removal of the surrounding cumulus and corona cells). This strategy allows not only precise injection of the oocytes, but also the assessment of their maturity, which is of critical importance for ICSI. Cumulus and corona cells are removed using a combination of enzymatic and mechanical procedures (20). Both the enzyme concentration and the duration of the exposure to the enzyme should be limited because they can result in...

Evaluation and Preparation of the Infertile Couple for In Vitro Fertilization

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. FSH levels also vary from cycle to cycle. A consistently elevated FSH predicts a poorer prognosis than a single elevated level with others being normal. The quality of ovarian stimulation is not improved when IVF is done in a cycle with a more normal FSH level (3). There is an agreement that women with a single elevated FSH level have a high cancellation rate, but studies conflict regarding the extent of reduction of pregnancy outcome (4,5). FSH levels are similar on days 2, 3, and 4. Women with premenstrual spotting should be advised to count the first day of full flow as day 1. It is the antral follicles that respond to stimulation. With a high-quality transvaginal ultrasound scan,...

Female Infertility Is Caused by Endocrine Malfunction and Abnormalities in the Reproductive Tract

Several treatments can alleviate infertility problems,- for example, some success has been achieved in hypothalamic disease with pulsatile administration of GnRH. When hy-pogonadotropism is the cause of infertility, sequential administration of FSH and hCG is a common treatment for inducing ovulation, although the risk of ovarian hyperstimulation and multiple ovulations is increased. Hyperpro-lactinemia can be treated surgically by removing the pituitary adenoma containing numerous lactotrophs (prolactin-secreting cells). It can also be treated pharmacologically with bromocriptine, a dopaminergic agonist that reduces the size and number of the lactotrophs and PRL secretion. Treatment with clomiphene, an antiestrogen that binds to and blocks estrogen receptors, can induce ovulation in women with endogenous estrogens in the normal range. Clomiphene reduces the negative feedback effects of estrogen and thus increases endogenous FSH and LH secretion. When reproductive tract lesions are...

In Vitro Fertilization The First Three Decades

International Federation of Fertility Societies, European Society of Human Reproduction and Embryology, Paris, France 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 wholly dependent on chance but which today is almost exclusively under human control. Today, nearly all forms of infertility can be treated by the various techniques of assisted reproduction, which are now responsible for the birth of around two million children worldwide. Although the origins of our medical knowledge of human reproduction are usually attributed to Hippocrates, so often described as the ''father of medicine, we do know that in the fifth century b.c. it was believed that both males and females each produced two seminal liquors, one stronger than the other a blend predominantly with the former...

Reproductive technologies help solve problems of infertility

The oldest and simplest reproductive technology is artificial insemination, which involves placing sperm in the appropriate part of the female's reproductive tract. This technique is useful if the male's sperm count is low, if his sperm lack motility, or if problems in the female's reproductive tract prevent the normal movement of sperm up to and through the oviducts. Artificial insemination is used widely in the production of domesticated animals such as cattle. The first successful ART was in vitro fertilization (IVF). In IVF, the mother is treated with hormones that stimulate many follicles in her ovaries to mature. Eggs are collected from these follicles, and sperm are collected from the father. Eggs and sperm are combined in a culture medium outside the body (in vitro, in glass), where fertilization takes place. The resulting embryos can be injected into the mother's uterus in the blastocyst stage or kept frozen for implantation later. The first test-tube baby resulting from IVF...

N974 Female infertility associated with male factors

Female infertility of other origin Female infertility, unspecified N98.0 Infection associated with artificial insemination N98.1 Hyperstimulation of ovaries N98.2 Complications of attempted introduction of fertilized ovum following in vitro fertilization Complications of artificial insemination by

Fertilization and Embryo Cleavage After ICSI

Fertilisation Polar Bodies Cleavage

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). Oocytes are considered normally fertilized when two individualized or fragmented polar bodies are present together with two clearly visible pronuclei (2-PN) that contain nucleoli (Fig. 3). The fertilization rate after ICSI is usually expressed per number of injected oocytes and ranges from 57 to 67 according to the sperm origin (45). As shown in Figure 3, abnormal fertilization may occur, reflected by one-pronuclear (1-PN) oocytes (about 3 of the injected oocytes) (45). These oocytes are likely to...

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, leading to fertilization failure, without the classical globozoospermia phenotype were also observed (57-60). The oocyte, in its turn, may also be responsible for complete fertilization failure, presumably because of deficiencies of the cyto-plasmic machinery capable of transducing the sperm-derived signals to adequate effector systems (57). Both the sperm-borne and the oocyte-borne abnormalities of oocyte activation can now be alleviated in the ICSI setting. Based on initial observations...

The Center For Human Reproduction

Embryology Andrology

A list of the principal pieces of equipment follows. It should be noted that there are consultants specific to the IVF field who specify equipment and do the laboratory layout, although the embryologist will have considerable input and may, in fact, take entire responsibility for the Medical Gases. There are a large number of medical gas cylinders within this suite. Even if general anesthesia is not used, there will be a need for centrally piped oxygen in the procedure rooms and the recovery room. The IVF culture area and micromanipulation area require vacuum A collection of fertility dolls from various cultures is interesting and relevant. Although not recommended for the waiting room, in a corridor, a large photo wall can be attractively created with photos of parents and their babies. Physicians in this specialty receive hundreds of photos from grateful patients. These range from poorly composed snapshots taken with disposable cameras to professionally posed photos with twins or...

Quality and Treatment of Sperm Used for ICSI

Probably the single most important justification for the development and application of ICSI for mouse colony management is the minimal requirement of sperm amenable to ICSI. Sperm from a male mouse that is fertile, subfertile, or infertile, including a male that either physically cannot mate or cannot produce normal sperm capable of fertilizing oocytes naturally either in vivo or in vitro, can be used successfully for ICSI. Sperm types that have been used successfully include epididymal and testicular sperm, which retains its motility after having been freshly harvested, following complete or hemicastration (Kimura and Yanagimachi 1995), and after having been recovered following cryopreservation ((Wakayama et al. 1998) K.C.K. Lloyd unpublished data). Normal offspring can also be obtained by injection of oocytes with spermatocytes (Kimura and Yanagi-machi 1995) and round spermatids (Sasagawa et al. 1998) from testis after artificial activation. It is this latter source that forms the...

In Vitro Fertilization of Endangered Species

Techniques of in vitro fertilization were first developed to aid couples who had not been able to conceive through normal sexual relations. In this technique, eggs were surgically removed from the mother and mixed with the father's sperm in the laboratory. If fertilization took place, one or more embryos were introduced into the mother's uterus in the hope that an embryo would implant and develop into a full-term infant. Almost immediately, these techniques were used in other animals, especially endangered species. It offered many advantages over natural reproduction. In pairs that showed little sexual interest in each other, eggs and sperm could be extracted, mixed in the laboratory, and viable embryos could be introduced into the female's uterus. Also, if there was little genetic diversity in a zoo population, sperm from a donor at another location could be sent and used. By the end of the twentieth century, in vitro fertilization was being coupled with surrogate motherhood. Here,...

Klinefelters Syndrome And Infertility

Previously, donated sperm or adoption were the only options for men with KS to have children, however, dramatic advances in infertility treatment for individuals with KS have recently occurred. Several reports of births resulting from intracytoplasmic sperm injection (ICSI) of sperm obtained from testicular biopsy specimens in patients with KS have been published (56-58). Although most patients with nonmosaic KS do not have sperm on testicular biopsy (59,60), this technique offers a chance at fertility for those who do. Unfortunately, analysis of spermatozoa obtained by testicular biopsy in individuals with KS has revealed an increased prevalence of sperm with an additional X chro mosome (61), higher rates of aneuploidy (62), and trisomy 21 (63), implying an increased risk of genetic abnormalities in offspring of individuals with KS born using ICSI. Additional selection of sperm from testicular biopsy specimens in individuals with KS may be required to prevent the transmission of...

Principles of ICSI

In ICSI, a single sperm, either intact or sperm head only, is mechanically inserted into the oocyte cytoplasm, bypassing all natural barriers to fertilization, including the cumulus layer, zona pellucida, and oolemma (plasma membrane). ICSI also precludes the natural incorporation of sperm membrane into the oolemma, as occurs during natural (non-assisted) fertilization. Sperm obtained from the epididymis do not need to be capacitated or acrosome-reacted when used for ICSI, although studies indicate that induction of the acrosome reaction may increase male pronuclear formation after ICSI (Kimura and Yanagimachi 1995). Mouse sperm has a hooked head that connects through the neck to a long tail, making it rather difficult to use for ICSI. Fortunately, only the sperm head is necessary for ICSI fertilization and embryo development in the mouse (Kuretake et al. 1996). In most mammals, including humans, centrioles and centrosomes located in the neck region are inherited paternally (Schatten...

Transgenesis by ICSI

Using sperm as a vehicle for delivering exogenous DNA into an egg is an interesting potential application for ICSI in the field of animal transgenesis and biotechnology. The ability of sperm to take up and bind exogenous DNA and then transfer it into an egg during fertilization was first described 30 years ago (Brackett et al. 1971) and was rediscovered in 1989 (Lavitrano et al. 1989). Studies have shown that mouse epididymal sperm can take up 1.5-4 x 106 DNA molecules (average 5 kB). The mechanism of DNA uptake and binding of sperm relies on an ionic interaction that takes place in the subacrosomal segment of the sperm head between DNA and a group of positively charged sperm membrane proteins. Sperm-bound DNA is rapidly internalized into nuclei in 60-65 of sperm (Lavitrano et al. 1992 Francolini et al. 1993 Zani et al. 1995). While the usefulness of this method applied to sperm to be used for transgenesis by IVF remains controversial, exogenous DNA can be delivered successfully into...

Infertility

Cystic fibrosis is associated with infertility in men. Up to 80 of men with congenital bilateral absence of the vas deferens have mutations in CFTR, although many of them have no obvious other symptoms. The most common of these mutations are AF508, G551D, G542stop, W1283stop, N1303K and

Conventional IVF

As compared with natural fertilization, sperm selection is less stringent in in vitro fertilization (IVF) because the fertilizing spermatozoon does not need to penetrate through the cervical mucus and to make its path along the female genital tract until it meets the oocyte. Hence, the probability of fertilization with less motile and less viable spermatozoa is higher. However, nonprogressively moving and immotile spermatozoa are still excluded as well as spermatozoa with defective function of binding sites for the zona pel-lucida and oolemma on their plasma membrane and those with abnormal acrosomal function.

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 morphology of the spermatozoa, but also include low sperm counts and impaired kinetic quality of the sperm cells. ICSI can also be used with spermatozoa from the epididymis or testis when there is an obstruction in the excretory ducts. Azoospermia caused by testicular failure can be treated by ICSI if enough spermatozoa can be retrieved in testicular tissue samples. Table 1 gives an overview of the current indications for ICSI. Table 1 Current Indications for Intracytoplasmic Sperm Injection...

ICSI Instrumentation

Despite the very successful and multiple applications of ICSI as an effective ART in mice, the complex nature of the procedure and specialized instrumentation required have been cited by some as making ICSI inaccessible to most workers in the field, with the exception of a few highly specialized laboratories. These are unfair assertions, as the instrumentation differs in only a few respects from that used in most transgenic laboratories today, and the procedure can be mastered with concerted daily practice and effort. The instrumentation needed to establish ICSI in the laboratory can be divided into two groups that required for preparation of the microtools, and that required for the ICSI procedure itself. A puller and microforge are necessary to prepare the microtools. The correct settings and use of these instruments are of critical importance in preparing good microinjection pipettes, which in turn is crucial to the ICSI procedure itself. These details are described in Sect. 2.5....

ICSI Procedure

Injection Needle For Oocyte Manipulation

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 (64). The micromanipulators allow three-dimensional manipulation (coarse and fine movements) of the holding and injection pipette on the left- and right-hand side, respectively. The microinjectors are used to either fix or release the oocyte with the holding pipette, or to aspirate and inject a spermatozoon with the injection The ICSI procedure involves the injection of a single motile spermatozoon into the oocyte. The procedure is carried out in a plastic microinjection dish containing...

Clinical Reproductive Endocrinology Stimulation Protocols

The first IVF baby was born as a result of an oocyte picked up in a natural cycle. However, the success rate of this protocol was very low, and the Monash group first reported large numbers of eggs and improved pregnancy rates using a stimulation protocol of clomiphene citrate and human meno-pausal gonadotropin (HMG) together (32). Several other regimens using these two medicines were subsequently reported. The common problems with these protocols were that endogenous gonadotropins led to premature luteinization in 30-40 of the cases, and, in others, ovulation occurred at an inconvenient time of the day. The major step in simplifying IVF induction of ovulation protocols and preventing these unwanted phenomena came with the introduction of GnRH agonists. GnRH analogs were created by a series of modifications in the GnRH molecule that led to the availability of new agonists and antagonists. The agonists initially enhance gonadotropin released from the pituitary but, with continuing...

Clomiphene Citrate and Fshhmg Protocol

This protocol used to be the preferred protocol in most IVF units prior to development of GnRH analogs. As mentioned, it was abandoned in our program as the dominant superovulatory regimen because of the high cancellation rate due to untimely LH surges and some reports of the possible adverse effect of clomiphene citrate on the endometrium. In our unit, this

Polycystic Ovarian Syndrome

PCOS is the most common cause of anovulatory infertility. This syndrome was first described by Stein and Leventhal in 1935. The PCOS Rotterdam Consensus Conference recently defined PCOS as a clinical syndrome comprising any two of the three features amenorrhea or oligomenorrhea clinical or biochemical hyperandrogenism, and bilateral polycystic ovaries on ultrasound (45). Although no single biochemical test is diagnostic of PCOS, most patients showed a characteristic ovarian ultrasonographic appearance, namely the presence of > 10 follicles between 2 and 10 mm in diameter. On the other hand, ultrasonographically identified polycystic ovaries are a common finding in apparently normal women, with a prevalence of Infertile women with PCOS represent a difficult therapeutic problem for assisted reproductive techniques because they have a higher incidence of OHSS (refer following section) (49). On transvaginal ultrasound scanning, the prevalence of polycystic ovaries has been reported to...

Oral Contraceptive Pretreatment

The role of GnRH antagonists in human IVF is discussed in Chapter 5. Oral contraceptive pre-treatment for ovarian stimulation in a GnRH agonist or a GnRH antagonist cycle has been recently investigated. Oral contraceptive scheduling of a GnRH agonist or a GnRH antagonist protocol results in fol-licular growth and hormone profile are similar to those observed in GnRH agonist protocols. The number of premature LH rises remains low. Similar numbers of oocytes and high-quality embryos are obtained. This is significant because the use of the oral contraceptive pre-treatment method significantly improves scheduling in a typical IVF program operating Monday to Friday. The greater convenience of oral contraceptive pre-treatment scheduling appears off-set by the need for longer stimulation protocols and more FSH than with a nonschedule regimen.

Ovarian Stimulation And Ovarian Cancer

Concern has been expressed that exposure to fertility drugs might be associated with a risk of ovarian cancer. In particular, pooled analysis of three case-controlled studies by Whittemore et al. (75) suggested an odds ratio of 2.8 (95 CI 1.3-6.1) for invasive ovarian cancer infertile women treated with fertility drugs compared with women with no diagnosis of infertility or infertility drug treatment. This study, suggesting an apparent trebling of risk in infertile women treated with fertility drugs, has been controversial and widely criticized for various biases in the study design. In 1994, Rossing et al. undertook a study that yielded the best data, to this time, examining the role of clomiphene citrate and ovarian cancer. Rossing et al. used record linkage with a population-based cancer register. They identified an increased incidence of ovarian cancer comprising both borderline malignant tumors and invasive disease. They found a standardized incidence rate (SIR) of 2.5 with 95...

Phase Ii Dosefinding Studies Single Dose Protocol

To determine the minimal effective dose, we conducted a dose-finding study. We compared the use of 2 and 3 mg to investigate the protection period,'' the time during which an LH surge is prevented after the antagonist administration. The IVF-ET results were strictly comparable between the two doses and the 2mg dose prevented LH surges for three days in all the patients. However, we observed that the suppression of LH tended to be reduced three days after the injection in the 2mg dose and an LH surge was observed four days after the 2mg Cetrorelix administration. The 3mg dose was therefore selected as a safer choice, as a ''protection period'' of at least four days can be obtained (14). No LH surge was observed in all the patients treated with the 3mg dose. In some patients, an LH rise (LH > 10IU L) was observed on the day of the antagonist administration. The Cetrorelix was able to prevent any further rise in LH, lowering immediately the LH levels and no surge was observed in these...

Multiple Dose Protocol

In all the studies presented, the multiple-dose protocol uses 0.25mg day of Cetrorelix or Ganirelix. To compare the antagonist multiple dose protocol (0.25mg day) to the GnRH-a in IVF cycles, the European Cetrorelix Study Group (21) published the results of an open randomized trial. They studied 188 patients treated with Cetrorelix and 85 patients treated with the long (Buserilin) agonist protocol both groups received hMG. The authors transferred embryos in 83.5 of Cetrorelix group versus 79 of Buserelin group. The clinical pregnancy rate was 22.3 and 25.9 per started cycle in the Cetrorelix and Buserilin groups, respectively these differences were not statistically significant. The duration of treatment with gonadotropins and the estradiol serum levels on the day of hCG were lower in the antagonist The European Ganirelix Study Group (22) also performed a controlled, multicentric, randomized trial to compare two treatment regimens for ovarian stimulation (multiple-dose antagonist vs....

GnRHnt In Mild Stimulation

Very few data are available on the use of CC-hMG rec-FSH and antagonists in IVF (29,30). Pregnancy rates appear satisfactory but a high incidence of LH surges was observed (31). It was demonstrated in an animal model that CC increases the sensitivity of the pituitary to GnRH and that the dose of antagonists might need to be increased when CC is used. A prospective study found a lower pregnancy rate in CC-hMG cycles and did not support the interest in this protocol (32). The revival of the natural cycle was proposed as the use of GnRH-nts could prevent premature LH surges. The objective of this regimen is to combine the possible prevention of an LH surge by the administration of the GnRH-nt and the simplicity of the natural cycle with minimal stimulation (Fig. 2). We investigated the administration of the Cetrorelix in the late follicular phase of minimally stimulated cycles in women of good prognosis. These patients had an age between 26 and 36 years old (mean 34.1 1.4), normal...

Indications Pcos Patients

Aspects of PCOS patients is the increased LH tone secretion. This group of patients is characterized by anovulation, and ovarian ovulation induction is usually performed using clomiphene citrate, FSH associated or not with GnRH agonists. The rationale for the use of GnRH-nt in PCOS patients is the fact that the LH FSH ratio will be decreased since LH secretion is more affected by the antagonist administration than FSH secretion (2). In IVF, another clear advantage is the reduced incidence of OHSS with the utilization of GnRH-nt. The use of GnRH-nt protocol allows also to induce the oocyte final maturation with GnRH agonist, to elicit an endogenous LH surge and, subsequently, decreasing the risk of OHSS (61). However, a large prospective trial is necessary to confirm these physiological hypotheses (see infra). Lubin et al. (62) described two case reports of PCOS patients treated with GnRH-nt before the treatment with GnRH agonist to induce ovulation. The patients showed a normalization...

The Need for LH Activity Adjunction

The need to counteract the dramatic decrease in LH observed in the GnRH-nts cycle has been proposed. No large prospective study is yet published comparing patients treated with GnRH-nt in which LH activity was added. LH activity could be added by the adjunction of rec-LH or by a small dose of hCG (70). It could also be obtained by using hMG. Profound suppression of LH was not found to be associated with lower pregnancy rates (71). No significant advantage was found by adding rec-LH on a systematic basis in GnRH-nts single and multiple-dose protocols (72,73). This remains to be confirmed in large published studies.

Programmation of GnRHnt Cycles

The programmation of GnRH-nt cycles is important to organize the work load of large IVF centers. Programmation could be obtained with progesterone administration in the late luteal phase or oral contraceptive pill (74). A more original way of programming the cycle was proposed by Fanchin et al. (75). The authors proposed luteal administration of estradiol in the late luteal phase as proposed by de Ziegler (76). The administration of estradiol prevents the FSH rise induced by the luteolysis and could synchronize the follicular cohort (75). Some authors (Kolibianakis et al., unpublished data) found lower pregnancy rates when an estro-progestative pill is used. The results of this study are not yet confirmed by other authors. In poor respon-ders, the use of contraceptive pills to program GnRH-nt cycle was found to be associated with increased cancellation rate (77).

Triggering Ovulation with GnRHa in GnRHnts Cycles

A recent study, comparing hCG, Lupron (0.2 mg), and Triptorelin (0.1 mg) to trigger ovulation in IVF patients treated with Ganirelix, found similar IVF-ET results between the three groups of patients (88). Other studies found lower pregnancy rates in patients treated with a combination of GnRH-nts and agonists (89). A small group of high responders were treated with a combination of GnRH-nt and agonists and no OHSS was observed in this preliminary report (90). As mentioned earlier, a study using Antide as GnRH-nt showed an altered luteal phase in cycles combining GnRH-nt and agonist (81). Some authors advocate that the luteal phase needs to be supported by both progesterone and estrogens (Itskowitz, personal communication). In a prospective randomized study, reduced pregnancy rate was found in GnRH-nt cycle in which GnRH-a was used to trigger ovulation, despite using progesterone and estradiol as luteal supplementation (91).

Perinatal Outcome of Pregnancy After GnRHnt for Ovarian Induction

Recently, two papers have been published on the perinatal outcome of IVF pregnancies obtained with GnRH-nt. One report followed 67 pregnant patients after ovarian induction with Ganirelix multiple-dose protocol (95). The miscarriage rate was 9 , and full data on perinatal outcome was obtained in 61 patients. The mean gestational age was 39.4 week for singleton pregnancies and 36.6 week for multiple pregnancies. A birth weight lower than 2500 g was present in 8.7 , one baby had a major congenital malformation, and seven minor malformations were reported in five infants. These results were not different from data available on IVF pregnancies.

Ultrasoundguided Oocyte Retrieval

The most visible use of ultrasound imaging in IVF has been the tremendous advance facilitated by transvaginal retrieval of oocytes (45-55). Oocyte retrieval was a procedure-limiting step when IVF was first done. Retrievals were done laparoscopically or using ultrasound guidance from transurethral, transvesicular, or transabdominal approaches (48,51,55-58). The advent of transvaginal transducers and concerted efforts to develop effective, accurate tracking of the needles used for follicle aspiration was probably the single most important step in making IVF as safe and effective as it is today (Fig. 3) (45,53-55,59-63). Retrieval of oocytes in IVF cycles is now routinely performed under TVUS guidance (49). An aspirating needle is introduced through a guide attached to a transvaginal probe and is inserted into first one ovary, then the other, via the vaginal fornices. Almost all aspiration needles now in common use have a small band of highly reflective surface near the tip of the needle...

Three Dimensional Imaging of the Endometrium

Three-dimensional (3D) ultrasonography first became available in the late 1990s and 3D is now a part of almost all high-end imaging systems. There are several methods used to provide 3D information and there are no studies comparing the same endpoints with different imaging systems (105). The prospects for predicting the probability of implantation in IVF programs have now extended into 3D exploration of endometrial receptivity (106). When endometrial volumes were compared among women who conceived and those who did not, pregnancy and implantation rates were significantly lower in women with volumes of less than 2mL, and no pregnancies were established when endometrial volumes were less than 1 mL. A contemporary study found no relationship between 3D volume of the endometrium and conception (106,107). Nor was a correlation found among estradiol levels, endometrial thickness, or endometrial volume, leading the authors to conclude that there was no predictive value for conception in...

Ultrasoundguided Embryo Transfer

There is a measure of controversy regarding the usefulness of ultrasonographic guidance during embryo transfer versus non-visually guided clinical touch (131,137,141,149-155). Some clinicians prefer to rely on ultrasound guidance for mock transfers in cycles before IVF and embryo transfer and clinical touch in the actual procedure, others use ultrasound guidance for all procedures, and still others make a decision regarding its use based on whether or not the transfer is likely to be classified as easy or difficult (137,144,155). Two recent meta-analyses and a subsequent randomized controlled trial have been interpreted to mean that transabdominal ultrasound guidance versus clinical touch for embryo transfer significantly increased the pregnancy rate, although the rates of miscarriage, ectopic pregnancy, and multiple pregnancy were not affected (154,156-158).

Introduction Laparoscopy

Before the era of clinical in vitro fertilization (IVF), oocytes were acquired by laparotomy (1). Aside from the escalating scientific interest in fertilizing and culturing human oocytes in vitro, the development of laparoscopic surgery also facilitated the use of IVF in treatment of infertile couples. At the end of 1960, the laparoscopic technique made it possible to retrieve human oocytes quite simply for both research and clinical purposes. The technique played a key role in the start of collaboration between Edwards, the scientist with the knowledge of how to fertilize and culture human oocytes in vitro, and Steptoe, a clinician mastering a technique that could be used to harvest oocytes in women with tubal infertility (2). Subsequently, laparoscopy became the technique of choice for oocyte aspiration during the first 10 years of this clinical IVF era. Different technical aspects surrounding this technique were published at that time by those groups involved in the early clinical...

Technical Aspects Equipment

There are today many different needles that are specially designed for oocyte aspiration. Most companies, like Cook Ltd., Swemed International Lab, and Wallas Ltd., which have specialized in equipment for IVF, offer different types of needles for oocyte retrievals. Our opinion after nearly 20 years of experience with TVOR and having tested most of the available needles on the market is that the sharpness of the needle is the most important factor. A sharp needle means less pain if the puncture is performed in analgesia. Furthermore, it is important that the surface of needle tip has Suction of follicular fluid can be performed either with a syringe or a suction pump creating the negative pressure needed. It was shown earlier that a negative pressure of 90-120 mmHg seems to be optimal for a good recovery and exerts no harm on the oocyte cumulus complex when aspirating mature follicles. However, aspirating immature oocytes from follicles of In conclusion, one should use a ready-to-use...

IVM of Oocytes from Women with PCOS

PCOS is a very heterogeneous syndrome, often first diagnosed when the patient presents complaining of infertility approximately 75 of these women suffer infertility due to anovulation. The majority of women with anovulation or oligo ovulation due to PCOS have menstrual irregularities, usually oligo- or amenorrhea, associated with clinical and or biochemical evidence of hyperandrogenism. In almost all these patients, ultrasonic scan of the ovaries typically reveals numerous antral follicles (28,29). Fertility treatments for women with PCOS include lifestyle management, administration of insulin-sensitizing agents, laparoscopic ovarian drilling, ovulation induction, ovarian stimulation, and IVF. As previously mentioned, this group of patients has an increased risk of severe OHSS from gonadotropin stimulation compared with women who have normal ovaries (5,30). The risk of multiple-follicle ovulation and subsequent multiple pregnancies is also of crucial importance (5,31). However, the...

IVM for High Responders to Gonadotropin Stimulation

Risk can be reduced by withholding the ovulation-inducing trigger of hCG (34). Thus, in conventional ovarian stimulation for IVF where there has been an over-response and there is a high chance of developing OHSS, the cycle would be cancelled. Immature oocyte retrieval followed by IVM and IVF may provide an alternative to cancellation of these cycles. Initially, one live birth was reported from immature oocytes collected from a patient at substantial risk of developing OHSS (35). More recently, Lim et al. (36) reported 17 patients with a high risk of developing OHSS during the course of their IVF cycles. Instead of canceling the cycles, they undertook immature oocyte collection followed by IVM. hCG was administered 36 hours before oocyte collection when the leading follicle had reached a mean diameter of 12-14 mm and indeed 11.6 of the oocytes had already reached the metaphase II stage at collection. Eight out of 17 (47.1 ) clinical pregnancies were achieved in this group of patients....

Priming with hCG and Pretreatment with FSH

We have shown that hCG priming prior to IVM oocyte collection increases the maturation rate of oocytes in vitro (27) and we administer 10,000 IU of hCG 36 hours prior to collection. A prospective, randomized controlled trial demonstrated no improvement in oocyte maturation rates with 20,000 IU of hCG compared with 10,000 IU of hCG therefore, there is no benefit from the higher dose (60).

Maturation In Vitro and Fertilization

Immature oocytes are incubated in a culture dish containing maturation medium. The maturation medium is supplemented with 75 mIU mL of FSH and LH. The oocytes are cultured at 37 C in an atmosphere of 5 carbon dioxide and 95 air with high humidity. Oocytes are checked for maturity 24 and 48 hours after culture. The oocytes are denuded of granulosa cells, and mature oocytes (detected by the presence of an extruded polar body) are fertilized by ICSI. ICSI is performed for in vitro matured oocytes because it reduces the risk of unexpected poor fertilization as compared with IVF. However, it has been demonstrated that ICSI may not always be essential for the fertilization of in vitro matured human oocytes collected from unstimulated ovaries when the sperm parameters are normal (61). After ICSI, the oocytes are transferred into 1 mL of IVF medium in a tissue culture dish. Fertilization is assessed 18 hours after ICSI by examining the oocytes for the appearance of two distinct pronuclei and...

Sperm Preparation Methods Simple Washing and Dilution

The sperm preparation method used for the first IVF cases involved dilution of the semen with culture medium (usually at 2-10 times the volume) and separation of the spermatozoa by centrifugation. After removal of the supernatant, the pellet is resuspended in another aliquot of culture medium. Repeat centrifugation, usually two or three times in total, is often used to ensure removal of contaminating seminal plasma. The centrifugation is usually performed at 200-300 g and it should certainly be performed at centrifugal forces less than 800 g (8). Advantages of this method are that it is the simplest and the least expensive to perform. One disadvantage of this technique is nonviable, and immotile spermatozoa as well as any leukocytes, squamous epithelial cells, or non-cellular debris that contaminated the original semen sample will still be present in the washed sample. Another disadvantage is the concern about potential damage caused by centrifugation. by significantly diminished...

Postseparation Treatment Of Spermatozoa Improvement of Motility and Sperm Function

The use of methylxanthine derivatives such as pentoxifylline for the stimulation of sperm functions, especially motility, is well known. Pentoxifylline is a nonspecific inhibitor of phosphodiesterase that has stimulatory effects on sperm motility and motion characteristics like sperm velocity or hyperactiv-ity. The stimulatory effect is attributed to increased intracellular levels of cAMP via inhibition of its breakdown by cAMP phosphodiesterase. Pentoxifylline is also reported to enhance the acrosome reaction (32) presumably due to the increasing levels of cAMP. The results of pentoxifylline treatment in assisted reproduction are equivocal. Depending on the conditions, especially the time of stimulation relative to the capacitative state of the spermatozoa and the concentration of pentoxifylline in the medium, overstimulation can result in a premature acrosome reaction (12). Thus, pentoxifylline tends to be used on a limited basis in IVF programs and some programs choose to use...

Outcome And Childrens Health

For all forms of assisted reproductive technology (ART), the most important outcome parameter is the health of the children born after ART. The birth of a healthy singleton birth has to be considered as the most important outcome parameter after ART (79). Even after several decades of ART practice, one has to realize that it is impossible to give an answer with regards to risks for pregnancy and birth complications for ovarian stimulation in view of timed intercourse and intrauterine insemination. Only in IVF and ICSI have enough data been collected to provide a valid estimation of the risks. Even then, there are limitations in the study design of IVF and ICSI follow-up which make it impossible to estimate whether it is the ART procedure or the underlying infertility of the treated couples that influence

Pregnancy Complications

Perinatal outcome of singletons born after IVF have been recently assessed in a meta-analysis (81). The study compared for the period 1978-2002, a cohort of 12,283 IVF and ICSI singletons to a control cohort of 1.9 million spontaneously conceived singletons, matched for maternal age and parity. In comparison with spontaneous conceptions, IVF and ICSI pregnancies were associated with significantly higher odds of each of the perinatal outcome parameters studied perinatal mortality, pre-term delivery, low birth weight, very low birth weight, and small gestational age. In the ART singletons, the prevalence was higher for early pre-term delivery, spontaneous pre-term delivery, placenta praevia, gestational diabetes, pre-eclampsia, and neonatal intensive care admission. IVF patients must be counseled about these adverse perinatal outcomes, and obstetricians should manage these pregnancies as high risk. A systematic review by Helmerhorst et al. of perinatal outcome of singletons and twins...

Possible Causes of Adverse Outcome

The genetics of the male and female partners may influence the outcome. It has been well established that there are more constitutional abnormal karyotypes in infertile males and females. Several studies also indicated that abnormal sperm have more chromosomal abnormalities. In a cohort of 1298 ICSI parents seen for genetic counseling, it was concluded that there was an increased genetic risk for 557 of these children (45). This increased risk was due to maternal or paternal age, chromosomal aberrations, monogenic or multifactorial disease, and consanguinity. Slightly less than 5 of infertile males and 1.5 of tested females had an abnormal karyotype. With regard to fetal karyotypes after ART, there are only systematic data available for ICSI (90). Results on 1586 fetal karyotypes indicated an increased risk related to the chromosomal anomalies in the parents. The majority of cases (17 out of 22) were paternally inherited. There were significantly more de novo anomalies (1.6 ), but the...

Intracytoplasmic Injection of Immature Male Germ Cells

The success of intracytoplasmic sperm injection (ICSI) in cases of extreme deficiency of sperm movement and morphology (12,13) has prompted attempts at using immature male germ cells for fertilization. These efforts were aimed at offering an ART option to men with azoospermia. Term pregnancies were achieved with epididymal and testicular spermatozoa as Immaturity of male germ cell nucleus (incomplete chromatin condensation) and cytoplasm (functional inferiority of developmentally relevant cytoplasmic components centriole and oocyte-activating factor) were suspected to decrease success rates of assisted reproduction with immature male germ cells (15). However, birth rates after fertilization with round spermatids from healthy experimental animals were close to those achieved with spermatozoa (16). Consequently, the relatively low clinical success rates reported after fertilization with round spermatids from infertile men appear to be related to the testicular pathology underlying...

Etiology And Diagnosis Of Abnormal Fertilization

Many abnormalities can be detected by noninvasive examination of zygote pronuclei (also refer to section Predictive Potential of Zygote Noninvasive Evaluation). However, pronuclear alterations are nonspecific and can be caused by both sperm-borne factors (50) and by those derived from the oocyte (51). Hence, the identification of the gamete of origin can only be evaluated indirectly, by putting together observations on zygotes and embryos from the present and the past assisted reproduction trials and analyzing them in the overall clinical context of each case. Sperm-derived, paternal factors are responsible both for abnormalities of fertilization and for defective preimplantation development. The most serious paternal deficiencies can cause complete fertilization failure or a failure of the male pronuclear development. Such conditions can be detected by diagnostic tests using heterologous ICSI with the patient's spermatozoa to mouse, hamster, rabbit, or bovine oocytes (52-54). As...

Cytoplasmic Component

Regardless of the different types of cytoplasmic inclusions, it has been observed that a deficiency in ooplasmic texture can also reduce reproductive success. Thus, oocytes with impaired fluidity of the cytoplasm, as assessed by the persistence of the injection funnel after ICSI, had a developmental disadvantage compared to MII gametes with regular viscosity (38). However, extensive cytoplasmic granularity is recognized as the most severe form of cytoplasmic texture anomaly since more than half of affected gametes show chromosomal abnormalities (39), which led to minimal rates of implantation (4.2 ) and clinical pregnancy (12.8 ).

Protein Macromolecules

Historically, the most commonly used protein source in human IVF and embryo culture was patient's serum, added to the culture medium at a concentration of 5 to 20 . In some programs, fetal cord serum was used in preference. The use of serum in embryo culture media has several inherent drawbacks the considerable expense and time required for its collection and processing (and screening of the fetal cord serum), the risk of infection to the laboratory staff, as well as the added stress to the patient. Serum contains many components which are poorly characterized. Furthermore, proteins in serum have macromolecules attached, such as hormones, vitamins and fatty acids, as well as chelated metal ions and pyrogens (184,185). As the concentration of such macromolecules and other serum components varies between patients and even within the menstrual cycle, it makes any comparison between batches of medium which contain serum almost impossible. Furthermore, serum from several groups of patients...

Incubator Incubation Chamber

As discussed above, for optimal development of human embryos in vitro it is important to maintain both the pH of the medium and temperature. The choice and use of incubators is therefore paramount for the success of an IVF program. Several studies have determined that embryo development in vitro is increased by restricting the opening of an incubator (105,274). Furthermore, the use of IR sensor incubators which restore CO2 levels within two to three minutes helps to alleviate the detrimental effect of repeated opening of the incubator (274). Alternatively, the use of modular incubator chambers (MICs) inside the incubator enables the gas phase that embryos are cultured in to remain constant. Mouse embryo development in MIC's at the same gas phase as the incubator (5 CO2 in air) was significantly increased compared to development in the main chamber of the incubator (105). The use of incubators that have multiple chambers within the single incubator chamber can also reduce fluctuations...

Multiple Pregnancies After ART

There is increasing evidence that the major outcome risk after all forms of ART is the occurrence of multiple pregnancies and births. This is the case for ovulation induction, ovarian hyperstimulation with or without intrauter-ine insemination, and IVF or ICSI (102). For IVF-ICSI, the number of children born has been estimated to be about two million. This positive observation is overshadowed by the fact that at least half of these children are not from singleton pregnancies. The occurrence of multiple IVF-ICSI pregnancies and births is, of course, due to the placement of more than one embryo. There is extensive evidence that multiple pregnancies and births generate more problems not only during pregnancy and delivery, but also later in life (102). Therefore, prevention of multiple ART gestation should be considered as a top priority for all infertility treatments. It is obvious that the practice of single embryo transfer may be the answer to this epidemic of multiple births. 3....

Flushing Follicles or

Flushing follicles or not has been debated for years. The rationale is that flushing of the follicle offers an advantage to the patient, with a larger number of oocytes being collected and therefore a higher potential for pregnancy. Kingsland et al. were the first to perform a randomized trial. They demonstrated that when performing transvaginal ultrasonically guided oocyte recovery, there were no significant differences in number of oocytes retrieved, fertilization rate, or pregnancy rate between those where flushing had been used as compared to no flushing. However, they could show that the operating time was significantly shortened in the non-flushing group (33). Similar results were later demonstrated in another prospective randomized study (34). In a recent publication, a study by Bagtharia and Haloob found that 40 of the oocytes were retrieved in the primary aspirate, whereas up to 82 of oocytes were retrieved with two flushes and 97 of oocytes were retrieved in up to four...

GnRH AgonistFSHLong Downregulation Protocol

This protocol (Fig. 2) is currently the protocol of choice for first cycle patients and previously normal or high responders in many IVF programs. A GnRH agonist Leuprolide, 1 mg SC, Buserelin 600 mg SC or Nafarelin acetate (Synarel) , 400 mg nasal spray, is given for 10-14 days before starting gonadotropin treatment (long-protocol), and may be commenced in the mid-luteal phase of the previous cycle or on day 2 of the cycle. We, in addition to other researcher found that pituitary suppression was more effective when the therapy commenced in the mid-luteal phase of the previous cycle Table 1 Follicle-Stimulating Hormone Starting Dose in In Vitro Fertilization Ovarian Stimulation Protocols Table 1 Follicle-Stimulating Hormone Starting Dose in In Vitro Fertilization Ovarian Stimulation Protocols The National Collaborating Centre for Women's and Children's Health have reviewed ovarian monitoring during gonadotropin therapy for IVF. NICE found that serum estradiol monitoring provided no...

Endometrial Thickness and Pattern for Assessing Endometrial Receptivity

Ovarian Follicle Aspiration Needle

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 > 9 mm but < 14 mm (75). In another study, no correlation was observed among endometrial pattern or thickness and estradiol levels, number of oocytes retrieved, or progesterone level on the day of embryo transfer however, the authors appeared to appreciate the pattern of the endometrium on the day of hCG administration, but stated that pattern assessment was of no value (78). In another IVF study, the endometrium on the day before embryo transfer was nearly 2 mm thicker in women who conceived (10.2mm) than in those who did not (8.6 mm) (79)....

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 before oocyte collection, when an extremely thin endometrium (i.e., < 4mm) is recorded on an ultrasound scan prior to the collection. We are currently investigating an alternative approach whereby the in vitro matured oocyte is vitrified when the endometrial lining is thin. The endometrium is then prepared in an artificial cycle and, once it reaches 8 mm, the oocytes are thawed, fertilized, and transferred. In an IVM treatment cycle, luteal support is started on the day that oocyte maturation is...

Testing Ovarian Reserve

Chance Pregnancy Age And Fsh

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 who make many eggs at this age, quantity does not make up for quality. By contrast, young women with limited ovarian reserve can have good success rates despite their limited cohort of eggs, because the eggs themselves are of high quality here quality matters more than quantity. The ramifications of these observations include the following Diminished ovarian reserve should not be used as an exclusionary criterion in young women, because overall they still have satisfactory pregnancy rates,...

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 been hampered by the routine inclusion of serum in human embryo culture media. Serum has the ability to both mask potential embryo toxins and suppress the beneficial effects of other medium components. In light of this, there has been considerable research into the development of serum-free embryo culture media. Such studies have been invaluable in our understanding of the embryo's requirements during the preimplantation period. These media were originally formulated to support the development of...

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 related to the degree of ovarian response to ovulation induction therapy, OHSS is probably an exaggeration of normal ovarian physiology. Part of the angiogenic response, which occurs in the follicle at the time of ovulation, is increased vascular permeability VEGF. VEGF stimulates endothelial cell mitogenesis and renders capillaries highly permeable to high-molecular-weight protein (59). VEGF has been identified in rat (60) and primate ovaries predominantly after the LH surge. Luteal-phase...

Perifollicular Blood Flow Doppler Indices

Serhal PF, Ranieri DM, Kinis A, et al. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod 1997 12 1267-1270. 5. Huey S, Abuhamad A, Barroso G, et al. Perifollicular blood flow Doppler indices but not follicular pO2, pCO2, or pH, predict oocyte developmental competence in in vitro fertilization. Fertil Steril 1999 72 707-712. 7. Barroso G, Barrionuevo M, Rao P, et al. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 1999 72 1024-1026. 11. Wang WH, Meng L, Hackett RJ, et al. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 2001 75 348-353. 13. Moon JH, Hyum CS, Lee SW, et al. Visualization of the metaphase II meiotic spindle in living human oocytes using the polyscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod 2003 18...

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 source used in the preparation of media. In contrast, George et al. (313) and Silverman et al. (314) With regards to alternative assays, such as hybridoma cell lines, although such cells in culture can be particularly sensitive to toxins in the medium, they are not embryonic cells and may therefore not detect potential embryo toxins. Although the mouse embryo bioassay does have a role in clinical IVF, at best it is only a test of the ability of the mouse embryo to develop. There is no...

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 decrease in the ovarian reserve, or number of follicles capable of being stimulated, is a primary reason for declining fertility. Similarly, the ovarian response to exogenous gonado-trophin stimulation also decreases, but the range of individual variation is extremely wide and it is well known that age is only a rough guesstimate of the ovarian reserve and hence the ovarian stimulation response. There are several tests of ovarian reserve'' that include clomiphene citrate challenge and the GnRH...

GnRH AgonistFSHShort Boost Protocol

Follicle Stimulation Protocol

This protocol (Fig. 3) is typically reserved for poor responders, namely patients with day 3 FSH > 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 The most important disadvantage of this protocol is the high P4 levels during the early follicular phase. This is most probably caused by rescue of the preceding corpus luteum. If the serum P4 is > 6nmol L in the late follicular phase, we recommend cancellation of the cycle. NICE guidelines confirm a decreased clinical pregnancy rate per cycle using the short protocol compared with the long GnRH agonist protocol. The relative cost of agonists in the various regimens required for IVF require further economic studies to ascertain whether there is any true difference in cost.

Spectral Doppler and Color Flow Doppler Ultrasonography

Ivf Embryo Transfer Ultrasound

Initially, attempts to determine if evaluation of blood flow in the uterine arteries could be useful were based on RI calculations to look for differences in uterine receptivity, where in a small series of women, no differences were found between women who conceived and those who did not (90). When the PI of uterine arteries was examined and data were grouped into low, medium, and high categories, no differences were found between cycles where women either conceived or did not however, no pregnancies were established in the women with high PI values (91). Elevated PI, as a measure of impedance to vascular flow in the uterine artery, was associated with a significantly lower pregnancy rate (92). However, no differences in uterine artery PI were observed between conception and non-conception cycles (93). A study assessing RI of the uterine arteries was inconclusive, except that absent or low diastolic flow was associated with failure to conceive (93). Uterine artery vascular impedance...

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, drawing the sample through a 21 gage needle into a syringe may help break up viscous globules. For men who are unable to collect semen by masturbation, nontoxic condoms are commercially available guidelines for their proper use should be strictly abided by patient and laboratory personnel. Ordinary contraceptive condoms must not be used (even those without spermicide) because of their sperm toxicity. Coitus inter-ruptus is also not recommended because of the risk of incomplete recovery and...

Fixed Versus Flexible Administration of the Antagonist

Multiple Dose Protocol Fixed Flexible

In the single dose, the 3 mg administration of Cetror-elix was proposed on day 7. We proposed a more flexible approach by adapting the moment of antagonist administration to ovarian response (Fig. 3) (11). The antagonist could be administered when the leading follicle reached 14 mm and or E2 reached 600pg ml. The same approach was later proposed by Ludwig et al. (57) in the multiple-dose protocol (Fig. 3). Some authors have predicted a lower pregnancy rate when the flexible approach was proposed (58). Data remain controversial on this question. A recent meta-analysis did not find differences in the pregnancy rates between the flexible and fixed approaches (59).

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.), IxaPrep (MediCult, Copenhagen, Denmark), and Enhance (Conception Technologies, San Diego, California, U.S.A.) are examples of silane-coated silica particle solutions that can be used for discontinuous gradients. These products are made isosmotic by the inclusion of polysucrose they have very low toxicity, are nonirritating, and are approved for human in vivo use. As with any product, it is important to follow the manufacturer's recommendation for proper use and application.

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 advantages of transvaginal follicle puncture is then lost. A good analgesic method for oocyte retrieval has to give a satisfactory pain relief with rapid onset, rapid recovery, and ease of administration and monitoring. It is also important that it is safe and has no toxic effect on the oocytes.

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 implantation rates decrease with increasing age. In women younger than 35 years, we have achieved a clinical pregnancy rate of 38 per oocyte retrieval and an implantation rate of 13 . In women between 36 and 40 years old, the clinical pregnancy rate is 21 per retrieval and the implantation rate 5 (Table 2). Based on more than 1000 IVM cycles in four centers performing IVM cycles with hCG priming before oocyte collection, the pregnancy rates reached 30-35 and the implantation rates 10-15 (63). Some...

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 tubes containing 0.9 saline with 2U mL of heparin. Because immature oocytes are enclosed in tightly packed cumulus cells, curettage of the follicle wall will dislodge the cumulus oocyte complex. In an immature oocyte collection, multiple needle punctures are needed. Because the aspiration pressure is low and a small-gauged needle is used, the bloodstained aspirate may often block the needle. Therefore, the needle is withdrawn from the vagina after aspirating a few follicles to flush and clear...

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 undergo this procedure in order to generate multiple embryos for genetic analysis. We have recently used IVM as an alternative for selected patients with PCO PCOS who require PGD so as to avoid the side effects of fertility-drug administration and avoid the risk of OHSS. We recently treated a 35-year-old patient with recurrent miscarriage who had been unsuccessfully treated with two IUI and two IVF cycles in Germany. We collected one MII and 14 GV oocytes and biopsied eight embryos generated....

Follicle Development and Oocyte Maturation In Vivo

Although it is clear that the LH surge triggers the resumption of meiosis in vivo, cumulus-oocyte complexes can be spontaneously induced to resume meiosis when they are released from follicles into culture in vitro. Therefore, the action of endocrine factors affecting oocyte maturation in vitro may be quite different from in vivo conditions. Immature oocytes, with or without surrounding cumulus cells, can be matured to the metaphase II stage however, the capacity of early embryonic development from the denuded oocytes is questionable. The beneficial effects of cumulus cells on early embryonic development have been reported in many species including humans (11). The actions of endocrine, paracrine, and autocrine factors that control oocyte maturation in vitro, either directly or indirectly, are mediated by the cumulus cells. Although FSH and LH play an important part in the development and maturation of preantral, antral, and preovulatory follicles in vivo, these gonadotropins may not...

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 frequency and amplitude of calcium oscillations is important not only for the immediate success of fertilization, but that it conditions further post-fertilization development during a relatively extended time period, up to the blastocyst stage (20-22). formation and internal structural differentiation of the male and the female pronucleus (11). Similar abnormalities occasionally occur spontaneously after conventional IVF or ICSI and are particularly frequent in some cases, suggesting the...

Monitoring the Course of Ovarian Stimulation

Tvus Ovulation Induction

Ovarian stimulation protocols vary tremendously and have evolved from fairly simplistic administration of exogenous hormones derived from urinary sources to quite sophisticated blends of gonadotrophin-releasing hormone (GnRH) analogs, recombinant follicle-stimulating hormone (FSH), luteinizing hormone (LH), and other compounds. The common denominator in all ovarian stimulation protocols is that ultrasonography is used to determine their effects on the ovaries of each patient. All the protocols have been designed to override the physiologic mechanism of selection of a single dominant follicle, obviate atresia in the cohort of follicles recruited into the follicular wave, and foster and sustain the development of many follicles to an imminently pre-ovulatory state so that properly matured oocytes may be retrieved for IVF. Ultrasonography is essential in determining the numbers and fates of individual follicles stimulated by exogenous gonadotrophins. Toward this end, the follicular...

The Function of Microtubule Organizing Centers

The microtubule organizing center (MTOC) is responsible for the initiation of microtubule organization in the meiotic and mitotic spindles. In humans, as in most other mammalian species, the oocyte MTOCs disappear after fertilization, and the sperm-derived centriole forms the basis for de novo development of the embryonic MTOC (25,26). If the oocyte is fertilized by a spermatozoon with a functionally abnormal centriole, microtubule organization is impaired and the function of the meiotic and mitotic spindles is defective (27,28). This functional abnormality can be revealed by a hetero-logous ICSI test using bovine oocytes and cytochemical visualization of microtubuli in newly formed asters with the use of antibodies against acetylated- and p-tubulins (28).

Conclusions And Future Considerations

IVM is an evolving technique which was initially indicated for patients with PCO because it avoids the necessity of ovarian stimulation, thus eliminating the risk of OHSS however, this technology should now be extended to other causes of infertility. IVM cycles require less monitoring and fewer clinic visits, lessening the burden on patients and further reducing costs which are already lower due to the lack of gonadotropin stimulation. IVM treatment could be offered as an alternative to IVF to all infertile patients who have a high number of antral follicles, and who are at increased risk of developing OHSS. For patients who are over-responding to ovarian stimulation, treatment can be converted to IVM when the follicles are still small. Poor responders may benefit from IVM treatment if they refuse oocyte donation, as they do not need to receive large doses of gonadotropins. For cancer patients seeking to preserve their fertility, IVM enables them to avoid hormonal stimulation and...

Multipledose

The Ganirelix Study Group (9) also investigated the minimal safe effective dose to achieve good IVF results. This study also showed that, during the multiple dose protocol with Ganirelix, the minimal effective dose was 0.25mg day, inhibiting the premature LH secretion without compromising IVF results in stimulated cycles with recombinant FSH. This group of patients, receiving Ganirelix 0.25mg day had the highest vital pregnancy rate per transfer (40.3 ) as the main clinical outcome if compared with the others doses (0.0625-2mg).

Single Dose Protocol

Moreover, a total of 18 patients of the Cetrorelix group (15.7 ) presented an LH rise (LH > 10 IU L) on the day of Cetrorelix injection. The administration of the Cetrorelix inhibited LH secretion. Four of them became pregnant (22.2 ). These interrupted LH rises seem to have no measurable deleterious effect in this study. Only one patient in the Triptor-elin group (2.8 ) experienced an LH surge. None of the 115 patients of the Cetrorelix group experienced an LH surge after the Cetrorelix administration. No LH surge has been reported so far within the four days following the single administration of 3 mg Cetrorelix. Triptorelin group. The E2 levels on the day of hCG were significantly lower in the Cetrorelix than in the Triptorelin group. The total number of follicles > 15 and < 17mm was higher in the Triptorelin group (5.0 3.9 vs. 3.4 2.6 CI 0.5-2.8). The long GnRH agonist protocol resulted in more oocytes and more embryos as already demonstrated in the literature when compared...

Poor Responders

Forty-two patients who are poor responders were divided into two groups for ICSI treatment (long GnRH agonist or Cetrorelix multiple-dose protocols) (65). The stimulation protocol also included, in some patients, clomiphene citrate associated with gonadotropins. Age, number of oocytes retrieved, number of fertilized oocytes, transferred embryos, score of embryo quality, and clinical pregnancy were not significantly different between the groups. A trend was observed in the pregnancy rates (14.28 for Cetrorelix vs. 9.52 for GnRH agonist treatment) but the difference was not significant. The authors discussed the sample size utilized. Probably, with an adequate power calculation this difference in terms of pregnancy rate can become statistically important. Other small studies have been published recently (67-69). Those studies did not find an improvement in pregnancy rates, but similar results were achieved.

Recommended Regimens

As we saw in this paper we still have questions on the best protocol to be used with GnRH-nts. From available literature, we can suggest that GnRH-nt can be used in a single- or multiple-dose regimen. The flexible or fixed approach appears also to give similar results. Programming the cycle can be obtained with OC pill or luteal estrogen. The latter lacks a prospective study to confirm its interest as opposite to the OC pill which is already used in many IVF cycles. The supplementation with LH activity is not supported by available scientific data. The supplementation of the luteal phase is recommended. If GnRH-nt appears to give comparable results in good responders, it should be studied in poor responders, patients with PCOS or IUI deserve further study.

Conclusion

In COH, the different studies presented have confirmed the efficacy of a single dose of 3mg of Cetrorelix to prevent premature LH surges when administered in the late follicular phase. The single-dose protocol is easy to use and assures patient compliance. When compared with the long protocol using a depot formula of Triptorelin, the IVF-ET results showed a shorter duration of treatment, less amount of hMG used, and a lower occurrence of OHSS in the group of patients treated with Cetrorelix. Clinical trials showed that the multiple-dose protocol using Cetrorelix or Ganirelix is effective and safe. A shorter duration of treatment, less amount of gona-dotropins, and a lower occurrence of OHSS was observed in the group of patients treated with Cetrorelix or Ganirelix. Single- and multiple-dose protocols have not yet been compared prospectively. Fixed day or flexible day schemes have not been compared either. The single dose is simple but requires monitoring of the cycle. The multiple...

Concluding Remarks

It seems strange to envision ART care without ultrasound imaging. The ability to see the effects of ovarian stimulation and optimize the protocol for individual patients, easy accessibility of the ovaries for oocyte retrieval, direct visualization of the endometrium at the time of embryo transfer, and embryo replacement under direct visual guidance are all easy to take for granted. Ultrasonography also provides direct visual access to all the events in human reproduction and allows us to elucidate biological phenomena approaching the natural state. One of the most joyous occasions we have in our work is when we are able to confirm pregnancy in our patients (Fig. 8). There is little that approaches the drama in the imaging suite when we can point out an embryo to our patients and listen to the heartbeat of their new life. When we combine the essential contributions to ART made by the dramatic advances made in the embryo laboratory with those made possible by advanced imaging...

Ultrasound

Clinical IVF started at the time when ultrasound imaging in obstetrics and gynecology developed rapidly. This was due to a rapid technical development within diagnostic ultrasound. Ultrasound was applied early for ovarian imaging and in some early studies it was elegantly shown that the technique could be used for monitoring follicular maturation (7,8). Furthermore, one of the Danish pioneers within diagnostic ultrasound proved earlier that the technique could be used safely and accurately for guidance of percutaneous puncture of cystic as well as homogenous tumors within different organs of the body (9). With the experience of ultrasound-guided puncture developed by Holm et al., it is not surprising that another Danish group lead by Lenz (10) demonstrated the first successful oocyte collection under the guidance of ultrasound. Parallel to Lenz et al., our own group in the beginning of 1980 started to use a similar technique for ultrasound-guided follicle aspiration (11). The...

Aspiration technique

The first two topics are interesting because almost all IVF clinics have their own methods of management. It is also very important to discuss updates related to complications within a technique. The longer a technique has been in use, the more information we will acquire about its safety.

Oocyte Donation

From immature oocytes retrieved and donated to a woman with premature ovarian failure (24). At our center, 12 oocyte donors (age 29 4) with high antral follicle counts (29.6 8.7) underwent immature oocyte collection without ovarian stimulation. A mean of 12.8 5.1 germinal vesicle (GV) oocytes were collected, 68 matured and underwent intracytoplasmic sperm injection (ICSI). A total of 47 embryos were transferred to 12 recipients and six (50 ) conceived, of which four have resulted in live births (Holzer H, Chian RC, Scharf E, Tan SL. IVM oocyte donors oocyte donation without ovarian stimulation, in preparation). Therefore, collecting immature oocytes from a donor's unstimulated ovaries in oocyte donation programs seems prudent and worthwhile.

Embryo Transfer

The fertilized oocytes are further cultured up to day 2 or 3, and then embryo transfer is performed. Assisted hatching is performed to avoid reduced implantation due to a hardened zona pellucida. When a large number of embryos have been formed, alternative approaches could be either an extended culture to the blastocyst stage or a double transfer (62). A double transfer is performed on day 2 or 3 and a blastocyst transfer on day 5 or 6. The embryo transfer technique is the same as that employed for conventional IVF.

Surgical

Quantities of sperm to be obtained and subsequently used for IVF, or even intrauterine insemination (IUI), and any excess sperm may be frozen for future use. Depending on operator skill, epididymal aspirates can be obtained with minimal red blood cell and non-germ cell contamination, making the isolation and selection of motile sperm quite easy. If large numbers of epididymal spermatozoa are obtained, then density gradient centrifugation (see below) is an effective method for preparing those spermatozoa for subsequent use.

Major Malformations

The question whether there is an increased risk for major congenital malformations after IVF or ICSI was recently reviewed in two meta-analysis (83,84). The meta-analysis by Hansen et al. (83) indicated an overall increase after IVF and ICSI. This was also the case when only singletons, IVF children or ICSI children were analyzed separately. The pooled odds ratio risk for major birth defects was 1.32 (confidence interval 1.20-1.45). A metaanalysis by Lie et al. (85) compared major malformations in 5935 ICSI children to 13,086 conventional IVF children. The relative risk for a major malformation after ICSI was 1.2 (95 CI 0.97-1.28). The meta-analysis by Rimm et al. (84) confirmed the higher risk of major malformations in IVF and ICSI children in comparison to spontaneously conceived children. There was no significant difference in the risk when IVF and ICSI were compared. A multicentric cohort study (86) of the physical health of 5-yr-old children conceived after ICSI (n 540), IVF (n...

Thomas Ebner

IVF-Unit, Women's General Hospital, Linz, Austria Taken together, viability of individual embryos is strongly correlated to optimal maturational steps in the ovary, adequate fertilization, progressive development through all pre-implantation stages, as well as subsequent implantation in the endometrium. Combining cytogenetical analysis morphological evaluation throughout preimplantation development (2), and embryo metabolism (3) the ability to select the most competent embryo out of a pool of concepti will further improve and definitely help to reach the ultimate goal in assisted reproduction, namely a healthy singleton delivery.

Nuclear Component

According to Gaulden (9), hypoxia is responsible for a reduction in metabolic activity as well as for a change in internal pH both of which are likely to affect organization and integrity of the meiotic metaphase spindle. This is supported by data from pre-antral follicle culture indicating that in vitro maturation at 5 oxygen tension (instead of 20 ) resulted in a significant reduction of gametes finishing nuclear maturation (10), e.g., characterized by a complete spindle absence. More interestingly, the rate of unaligned However, absence of a birefringent spindle does not predict fertilization failure and developmental arrest. In fact, it has been found that human oocytes with a polar body but without birefringent spindle may still be at telophase I or prometaphase I stage (18). Thus, precocious intracytoplasmic sperm injection (ICSI) in human prometaphase I oocytes with unaligned chromosomes may be one reason why oocytes without a birefringent metaphase II spindle have a...

The Zygote

Normal fertilization follows a defined course of events, although the timing of these events may vary considerably (for more details, refer to Chapter 11). Either direct deposition (ICSI) or active propulsion conventional in vitro fertilization (IVF) ensures presence of a spermatozoon in the cytoplasm. Its head decondenses in the ooplasm prior to the extrusion of the second polar body. The male pronucleus appears in the center of the oocyte

Ions

The ionic basis of culture media used for clinical IVF varies markedly (Table 4). Surprisingly, relatively little is known about the role of ions during preimplantation embryo development. The ionic composition of oviduct fluid from the human and mouse has been sampled by micropuncture and analyzed using an electron probe (Table 4) (30,44-46). Mammalian oviduct fluid is characterized by high potassium and chloride concentrations and a high overall osmolality (44,45). Interestingly, high osmolality balanced salt solutions with added carbohydrates as energy sources do not support high levels of embryo development in vitro (47,48). Studies on the effect of magnesium and calcium in the medium for the development of 2-cell mouse embryos in culture determined that magnesium was not essential for development to the blastocyst stage however, calcium is essential for embryos to undergo compaction in vitro (51,60). More recently, the effects of extracellular magnesium and calcium levels on the...

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