Manual In Vitro Fertilization: The A.R.T Of Making Babies (3rd Edition)

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  1. Assisted Reproductive Technology - Embryology
  2. Assisted Reproductive Technology Surveillance — United States, 2015
  3. In Vitro Fertilization: The A.R.T. of Making Babies (Assisted Reproductive Technology)
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However, tubal transfer was not universally applicable since patients with dense pelvic adhesions or tubal occlusion could not be treated, and patients with male factor infertility could not be observed for fertilization success or treated effectively. Subsequently, the technique of transferring laparoscopically retrieved oocytes fertilized in vitro at the pronuclear stage into the fallopian tube via a second laparoscopy was introduced.

This procedure was known as zygote intrafallopian transfer ZIFT and it allowed the confirmation of fertilization but maintained some of the theoretical benefits of GIFT Hamori et al However, the use of two laparoscopies, one for oocyte retrieval and the other for zygote transfer, was a major limitation of this approach. As the use of assisted reproductive treatments expanded from tubal infertility to include ovulation disorders, male factor infertility and decreased ovarian reserve, the number of ART cycles dramatically increased.

Assisted Reproductive Technology - Embryology

The pioneering work reported by Steptoe and Edwards had required a surgical approach using laparoscopy. However, the necessity for general anesthesia and its attendant risks, along with the high overhead expense of using operating rooms provided the impetus to develop more efficient nonsurgical oocyte retrieval techniques. Improvements in ultrasonography during the s catalyzed the evolution of modern outpatient oocyte retrieval. Using transabdominal ultrasound guidance, various methods of oocyte retrieval included percutaneous Lenz and Lauritsen , transvesical Lenz et al ; Lenz and Lauritsen , per-urethral Parsons et al , and transvaginal follicle aspiration Dellenbach et al Further refinements in ultrasound transducers led to the use of transvaginal ultrasound guided transvaginal follicle aspiration Figure 3.

First reported in , this oocyte retrieval technique quickly became the procedure of choice due to better visualization, finer control, and less patient discomfort compared with other available methods Wikland et al Obviating the need for laparoscopy decreased the number of personnel, time and procedure expense, reduced the risks of surgery and general anesthesia, and provided greater patient acceptance. IVF cases transitioned from 1—2 hours of hospital-based operating room time to 10—15 minute procedures that can be performed in an office setting. Schematic of modern outpatient in vitro fertilization setup.

Reproduced with permission from Cook Group Incorporated. Ovum aspiration diagram [online]. Accessed on 2 January Multiple studies in the early s comparing IVF, ZIFT, and GIFT often showed conflicting results due to multiple confounding variables inherent to different patient-selection criteria and the wide variation of practice used from clinic to clinic.

Nevertheless, potential enhancements in outcomes of GIFT and ZIFT were marginalized by the reliance on invasive surgery, especially as IVF evolved into less expensive and minimally invasive ultrasound guided aspirations. As the pregnancy rate of IVF improved over the last decade from As IVF became more commonplace in the treatment of female infertility, male infertility remained a limiting factor to overall success.

Conventional IVF was much less effective when semen parameters were below the reference values for concentration oligozoospermia , motility asthenozoospermia , and morphology teratozoopermia , resulting in significantly lower fertilization rates and fewer embryos available for transfer. Furthermore, azoospermic males were completely devoid of treatment options.

Several procedures were developed in the late s to improve treatment fertilization problems. The first technique developed was partial zona dissection PZD where a small opening was made in the zona pellucida in hopes of facilitating sperm entry into the oolemma. Another technique introduced was subzonal insemination SUZI where a few motile sperm were microinjected through the perivitelline space. Palermo and Van Steirteghem introduced a novel procedure called intracytoplasmic sperm injection ICSI , where a single spermatozoon was microinjected into the oocyte after passage through the zona pellucida and the membranes of the oocyte Palermo et al ICSI not only overcame the fertilization barrier presented by oligo-, astheno-, and teratozoospermia, it also created a new possibility for azoospermic men to achieve fertility.

While Temple-Smith and colleagues reported the first pregnancy by microepididymal sperm aspiration MESA in a patient with secondary obstructive azoospermia in , epididymal sperm motility was generally low and outcomes were poor compared with that of ejaculated sperm Temple-Smith et al The advent of ICSI markedly improved fertilization rates with epididymal sperm, making the combination of MESA—ICSI widely-used procedures for patients with congenital bilateral absence of vas deferens or secondary obstructive azoospermia. Subsequently, modified percutaneous sperm aspiration PESA performed with needle aspiration of the epididymis through a 1 cm scrotal incision, was developed as an alternative to MESA which required unilateral hemiscrototomy to allow dissection, exploration, and aspiration of the epididymis with an operating microscope Shrivastav et al When there are no motile sperm in the ejaculate or in the proximal epididymis, sperm can be extracted directly from testicular tissue by either blind needle puncture or open tissue excision.

Craft et al reported successful ICSI fertilization with testicular sperm in , but no pregnancy occurred. The first successful pregnancy was reported in that year Schoysman et al Pregnancies have even been achieved in conditions such as Klinefelter syndrome, generally associated with germ cell atrophy and fibrotic, hyalinized seminiferous tubules Staessen et al To date, there have been 39 reports of healthy children born after ICSI procedures with spermatozoa retrieved from nonmosaic Klinefelter patients since the first pregnancy in Denschlag et al As we enter the second decade using ICSI, debates continue on the implications of altering the process of natural selection.

Inheritable causes of male fertility such as constitutional chromosomal aberrations, cystic fibrosis transmembrane conductance regulator gene mutation, or AZF deletion on the Y chromosome may be inadvertently transmitted via ICSI. In addition, potential damage to the cytoplasmic organelles of oocytes inflicted by the procedure may increase the risk of health problems in children.

In a case series of ICSI established pregnancies, prenatal diagnosis showed a significantly higher percentage of de novo sex and autosomal chromosomal abnormalities 2. In addition, Hansen et al found the prevalence of major birth defects in infants conceived after ICSI, as well as IVF, were doubled compared with infants conceived naturally. Furthermore, a multi-center study with a five year follow-up period also showed a significantly increased risk of malformation in children born after ICSI compared with that of naturally conceived children, particularly in the genitourinary system of male children Bonduelle et al Hence, the current available evidence suggests the increased risk of congenital malformation associated with IVF is not attributable to ICSI, per se, and may be related to other factors such as patient characteristics, ovarian stimulation protocols, or embryo culture environment.

Assessment of the developmental outcome of children at 24—28 months of age by Bayley Scale shows no significant differences between the ICSI and IVF group Bonduelle et al In addition, motor and cognitive development are equivalent among IVF-conceived, ICSI conceived, and naturally conceived children at the age of five Ponjaert-Kristoffersen et al Nevertheless, there is evidence for increased incidence of otherwise rare and sporadic imprinting disorders such as Beckwith-Wiedeman and Angelman syndrome in ICSI children, possibly due to disruption of methylation in the maternal genome or early embryo by the procedure Sutcliffe et al ; Staessen et al ; Cox et al This implies that the embryo culture condition alone may alter DNA methylation and imprinting, as has been shown by in vitro mouse embryo studies Khosla et al Interestingly, Ludwig et al found a similar increase in the incidence of Angelman syndrome between children born to subfertile couples whose time to natural conception was greater than two years and children born to subfertile couples who underwent ART, regardless of ICSI.

These findings suggest that imprinting defects and subfertility may have a common cause, and superovulation with ART in this population may further increase the risk of conceiving children with imprinting defect. Due to the overall rarity of these syndromes, large-scale systematic studies are needed to clarify the link between genomic imprinting defects and ART as well as to establish the exact biological basis of any such link.

Assisted Reproductive Technology Surveillance — United States, 2015

Prior to , options to prevent transmission of genetic defects were limited to performing chorionic villus sampling or amniocentesis and offering abortion if the fetus were found to be affected. The 3 to 5 day window between oocyte fertilization and embryo transfer provided a new opportunity to delineate which embryos are unaffected by a specific single gene disorder or chromosomal imbalance prior to transfer into the uterus.

The first clinical application of this procedure called preimplantation genetic diagnosis PGD was used in to prevent the transmission of two X-linked conditions: adrenoleukodystrophy and X-linked mental retardation Handyside et al These embryos were biopsied on day 3 in vitro at the six to ten cell stages by first drilling a hole into the zona pellucida with acid Tyrodes and then aspirating blastomeres with micropipettes for analysis. Cells not containing the amplification were presumed as female, and the embryos of origin were transferred back into the uterus to prevent transmission of the X-linked diseases.

The first application of PGD resulted in two sets of healthy female twins Handyside et al Biopsies of polar bodies during meiosis rather than blastomeres during the cleavage stage were used in some of these cases. Although this approach may be less disruptive to the embryo, it only allowed analyses of maternal DNA and hence had limited clinical applications. In the last 15 years, the number of inherited disorders diagnosed at the preimplantation stage has expanded to over 40 diseases, and the advent of multiplex-PCR which simultaneously amplifies several DNA fragments in a single reaction greatly improved the accuracy of the analyses Kuliev et al In additional to single gene mutations, chromosomal screening represents another application of PGD, in an attempt to decrease the risk of transferring aneuploid embryos.

Initial techniques to identify X- and Y-chromosomes using PCR was quickly supplanted by fluorescence in situ hybridization FISH due to its speed and capacity to identify multiple chromosomes simultaneously Delhanty et al ; Munne et al ; Griffin et al Today, FISH remains the primary method for routine aneuploidy screening but has expanded up to 9 chromosomes per embryo-X, Y, 13, 15, 16, 17, 18, 21, and Aneuploidy screening AS is currently offered to various patient groups including women of advanced reproductive age, carriers of chromosomal rearrangements, and patients with history of recurrent pregnancy losses or unexplained repeated IVF failures Munne and Wells Evidence supporting the benefit of aneuploidy screening by PGD in the abovementioned patient groups remains equivocal.

A recent meta-analysis also failed to show significant increases in pregnancy rates using PGD for aneuploidy screening Twisk et al Since then, similar approaches to obtain stem cell from siblings for transplantation in couples with an affected offspring have been used for acute lymphoblastic leukemia, thalasemia, and Wiscott-Aldrich Kahraman et al Contemporary investigative efforts continue to focus on providing even more couples the opportunity to have healthy children. One such group is women diagnosed with cancer who often sustain partial or total loss of their fertility following cancer therapy.

While embryo cryopreservation prior to initiating potentially gonadotoxic treatment is the most reliable method to preserve fertility, absence of a male partner or desire not to use donor sperm often precludes this approach. In such cases, oocyte cryopreservation may provide an alternative. To date, there have been approximately pregnancies and 50 live-births from cryopreserved oocytes since it was first reported twenty years ago Van der Elst The large size, high water content, and chromosomal arrangement along the meiotic spindle render metaphase-II oocytes extremely vulnerable to the intracellular ice formation during the freezing or thawing process.

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In addition, hardening of zona pellucida can interfere with normal fertilization process. In terms of outcome, the incidence of chromosomal abnormalities in human embryos obtained from cryopreserved oocytes is similar to that of control embryos using FISH Cobo et al In addition, limited data based on the outcomes of 17 children resulting from cryopreserved oocytes showed no increased incidence of abnormal karyotype, preterm delivery, low birth weight, birth defects, intellectual or developmental deficits by the age of three Winslow et al Vitrification uses high concentration of cryoprotectants to solidify the cell into a glass-like state without the formation of ice.

Based on a small study, post-thaw survival rates and pregnancy rate of this approach were The other alternative of freezing oocytes at the germinal vesicle stage allows higher survival rates than those frozen at the metaphase II stage Boiso et al However, germinal vesicles need in vitro maturation IVM to become mature oocytes, and the inefficiency of current IVM protocol nullifies the improvement in survival rate, leading to equivalent final yield of mature oocytes as compared with that of freezing metaphase II oocytes.

At this time, both nascent protocols are continuing to evolve and lack long term outcome data. Despite the fact cryopreservation of oocytes obviates the immediate need for sperm, the inability to delay initiation of chemotherapy or the presence of estrogen sensitive malignancies precludes the use of ovarian stimulation and hence oocyte cryopreservation in many clinical scenarios.

Ovarian tissue cryopreservation, achieved by biopsying and cryopreserving ovarian cortex containing primordial follicles, followed by thawing and transplanting the autograft after completion of cancer treatment offers a potential solution in those circumstances.

Transplantation can be either orthotopic in close proximity to the infundibulo-pelvic ligament or heterotopic ie, forearm or abdomen. To date, transient restoration of endocrine function has been reported with both approaches. Embryo transfer resulting from stimulation of cryopreserved ovarian heterotopically grafted beneath the abdominal skin did not result in pregnancy. During in vitro fertilization IVF , eggs and sperm are brought together in a laboratory glass dish to allow the sperm to fertilize an egg. With IVF, you can use any combination of your own eggs and sperm and donor eggs and sperm.

After IVF, one or more fertilized eggs are placed in the uterus. Ovulation and egg retrieval. To prepare for an assisted reproductive procedure using your own eggs, you will require hormone treatment to control your egg production ovulation. This is done to prevent unpredictable ovulation, which would make it necessary to cancel that in vitro attempt.

This is typically done using one of two similar types of gonadotropin-releasing hormone analogue GnRH agonist or GnRH antagonist. The following are two examples of how ovulation can be controlled:. After the first week, your doctor checks your blood estrogen levels and uses ultrasound to see whether eggs are maturing in the follicles. During the second week, your dosage may change based on test results. And you are monitored frequently with transvaginal ultrasound and blood tests.

If follicles fully develop, you are given a human chorionic gonadotropin hCG injection to stimulate the follicles to mature. The mature eggs are collected 34 to 36 hours later by needle aspiration guided by ultrasound. You will usually have pain medicine and sedation for this procedure. Sperm collection. Sperm are collected by means of masturbation or by taking sperm from a testicle through a small incision.

This procedure is done when a blockage prevents sperm from being ejaculated or when there is a problem with sperm development. Sperm may have been collected and frozen at an earlier time.


Then the sperm are thawed on the day the eggs are collected. Fertilization and embryo transfer. The eggs and sperm are placed in a glass dish and incubated with careful temperature, atmospheric, and infection control for 48 to hours. About 2 to 5 days after fertilization, the best fertilized eggs are selected.

In Vitro Fertilization: The A.R.T. of Making Babies (Assisted Reproductive Technology)

One to three are placed in the uterus using a thin flexible tube catheter that is inserted through the cervix. Those remaining may be frozen cryopreserved for future attempts. Pregnancy and birth. Any embryos that implant in the uterus may then result in pregnancy and birth of one or more infants. Overall, in vitro fertilization IVF -related injections, monitoring, and procedures are emotionally and physically demanding of the female partner. Superovulation with hormones requires regular blood tests, daily injections some of which are quite painful , frequent monitoring by your doctor, and harvesting of eggs.

These procedures are done on an outpatient basis and require only a short recovery time. You may have cramping during the procedure. You may be advised to avoid strenuous activities for the remainder of the day or to be on bed rest for a few days, depending on your condition and your doctor's recommendation. IVF can be done using donor eggs for women who cannot produce their own eggs due to advanced age or other causes.

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The number of women who have babies after in vitro fertilization varies, depending on many different things. Cause of infertility. Infertility can be caused by problems with the woman's or the man's reproductive system. Some of these causes can include problems with the fallopian tubes, with ovulation , or with the sperm.

Pregnancy history. A woman who has already had a live birth is more likely to have a successful ART procedure than a woman who hasn't given birth before. This "previous birth advantage" gradually narrows as women age from their early 30s to their 40s. The most common one is a long GnRH-Agonist Lupron protocol where the secretion of gonadotropin hormones is suppressed in order to prevent premature ovulation.

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Once optimal suppression is achieved, the next step is the recruitment of multiple follicles by daily injections of gonadotropins. Ultrasound imaging and hormone assessments are used to monitor follicular development. When the lead follicles have reached the appropriate size, the final maturation of eggs is completed by HCG administration.

Egg retrieval is scheduled 34 - 36 hours after HCG injection. Egg retrieval is performed in a surgical suite under intravenous sedation. Ovarian follicles are aspirated using a needle guided by trans-vaginal ultrasonography. Follicular fluids are scanned by the embryologist to locate all available eggs. The eggs are placed in a special media and cultured in an incubator until insemination. There are several criteria used to assess the quality of the embryo. This is especially important when trying to decide which embryos to choose for embryo transfer.

Early in the morning on the day of your transfer, the embryos are evaluated and photographed by the embryologist. The embryologist and your physician will decide, based on the rate of development and appearance of the embryos, which and how many embryos are recommended to be transferred. Typically, embryos are transferred at the cleavage stage day 3 after oocyte retrieval or at the blastocyst stage day 5.

In the lab, a grading system is used to asses the quality of the embryos.

When analyzing these embryos, we not only look at the number of cells but also how symmetrical they are and whether there is any fragmentation. Fragmentation occurs when the cells divide unevenly, resulting in cell-like structures which crowd the embryo. No fragmentation is preferable but some is acceptable. In our lab, we classify embryos into grades 1 through 4. Grade 4 represents the best quality embryos. Day 5 embryos are called blastocyst embryos. At this stage, the embryos have increased in size and are even more developed. They resemble a ball of cells with fluid inside.