Continuing Education Activity

Assisted reproductive technologies (ART) are used to aid in achieving pregnancy conception in individuals who are having difficulty doing so spontaneously. This article reviews current assisted reproductive technology techniques, including indications for use, recommended techniques, common complications, and the importance of a coordinated interprofessional team in the reproductive medicine field.

Objectives:

• Review indications and relative contraindications of ART.
• Identify relevant female pelvic anatomy for ART.
• Discuss evidence-based techniques for in vitro fertilization and associated procedures.
• Outline complications of ART and subsequent management.

Introduction

Assisted reproductive technologies (ART), by the American Center for Disease Control (CDC) definition, are any fertility-related treatments in which eggs or embryos are manipulated. Procedures where only sperm are manipulated, such as intrauterine inseminations, are not considered under this definition. Additionally, procedures in which ovarian stimulation is performed without a plan for egg retrieval are also excluded from the definition.  

The first successful in vitro fertilization (IVF) treatment in humans was performed in 1978 in England – a woman had an unstimulated menstrual cycle, and physicians performed a laparoscopic retrieval of a single oocyte from the ovary. The oocyte was then fertilized in vitro and subsequently transferred as an embryo into her uterus.[1] 

Since that time, IVF technology has developed and expanded in access worldwide. This review will discuss current techniques in assisted reproductive technologies and discuss indications and associated risks. 

IVF is by far the most common ART procedure performed and will be chiefly reviewed along with associated techniques such as cryopreservation and intracytoplasmic sperm injection (ICSI).

Anatomy and Physiology

The crucial components of the female anatomy in understanding ART are the ovaries, fallopian tubes, and uterus. The ovaries are the third component of the hypothalamic-pituitary-ovarian axis (HPO), which is a complex feedback loop that controls the female menstrual cycle. The ovaries are the female gonads, paired oval-shaped structures that embryologically derive from the mesonephric ridge before descending in the pelvis. The ovaries are where oocytes mature and develop. Production of estradiol and progesterone also derive from the ovary. The ovaries have two peritoneal attachments – the ovarian ligament and the suspensory ligament of the ovary. The ovarian ligament attaches the ovary to the uterus. The suspensory ligament of the ovary attaches the ovary to the pelvic sidewall and contains the neurovascular supply to the ovary. Understanding this anatomy is important in understanding the transvaginal approach to oocyte retrieval. 

The uterus responds to the fluctuating hormones produced by the HPO axis. It lies in the pelvis, between the bladder and rectum. It consists of the corpus (the body of the uterus) and the cervix, which connects the uterus to the vagina. The uterine body consists of three layers – the perimetrium, myometrium, and endometrium. The endometrium is composed of two layers, the functionality, and the basalis. The functionalis layer undergoes cyclical hormonal changes to prepare for implantation with each menstrual cycle. Increasing estrogen levels during the follicular phase of the menstrual cycle results in endometrial glandular proliferation. During the luteal phase, progesterone levels rise, causing the endometrium to undergo secretory changes. If an embryo does not implant during the cycle, estrogen and progesterone levels fall, resulting in degradation of the functionalis layer, which then sloughs off with menses.[2]

The fallopian tubes are muscular tubes that extend on both sides laterally from the uterus towards the ovaries.  They aid in the transfer of the ovum to the uterus, often with fertilization happening within the tube itself. The tubes aid in this transfer by sweeping up the ovum at their fimbriated ends, with smooth muscle contractions and ciliated columnar epithelial cells transferring the ovum or embryo to the uterus where it can implant if fertilized.

Indications

Assisted reproductive technologies are most frequently performed secondary to infertility. In patients with tubal factor infertility, IVF directly bypasses the fallopian tubes. Other infertility etiologies in which IVF is employed include male factor infertility, diminished ovarian reserve, ovarian failure (with donor eggs), ovulatory dysfunction, and unexplained infertility.[3] In patients for whom pregnancy is relatively contraindicated (discussed below) or with uterine factor infertility, IVF can be used with a gestational carrier. 

IVF is also used outside of infertility settings. It can be used in patients desiring preimplantation genetic testing before conception (such as those known to be carriers of certain genetic disorders), fertility preservation, such as prior to gonadotoxic therapy, or in patients desiring to delay childbearing. These women can opt to freeze their eggs or embryos if they are in a stable relationship.[4]

Contraindications

Before initiation of ART, maternal risks of the ART techniques and pregnancy itself are discussed with the patient. Certain maternal conditions, particularly cardiopulmonary conditions such as pulmonary hypertension and heart failure, are relatively contraindicated conditions for pregnancy. Pre-conception counseling and evaluation to screen for such conditions should be conducted. Even in such patients, gestational carrier options can be utilized.[5]

Equipment

Based on American Society of Reproductive Medicine (ASRM) guidelines for embryology/andrology labs, the following basic equipment is required for the management of oocytes and embryos:[6]  

• Incubator 
• Microscope suitable for handling and micromanipulation of oocytes and embryos 
• pH and temperature monitoring and maintenance devices 
• Warming blocks 
• Laser for biopsy of embryos 
• Cryopreservation equipment (liquid nitrogen tanks) 
• Laboratory centrifuge 
• Laminar flow hood 
• Culture media 
• Refrigerator 
• Air filtration system

Personnel

Per ASRM, minimum personnel to appropriately offer assisted reproductive technologies includes a medical director, a physician licensed in reproductive endocrinology and infertility, a physician with male reproduction expertise, nursing with training in reproductive medicine and ART, an embryology laboratory director, an andrologist with laboratory procedure experience, laboratory personnel to perform the necessary hormone assays, an individual with experience in gynecologic ultrasound (physician, technician or nurse), a mental health professional with fertility counseling experience, and a genetic counselor.[7]

Preparation

 Preparation for ART procedures largely involves the evaluation and workup for etiologies of infertility. Infertility is defined as failure to achieve pregnancy after at least one year of unprotected intercourse. Infertility evaluation can also be initiated at six months of failure to achieve pregnancy in women over 35 or in cases where there are known possible barriers, such as known uterine or tubal disease or male infertility.[8] Initial comprehensive history taking includes menstrual history, pregnancy history, infertility duration, prior infertility treatments, past medical and surgical history, family history, and social and environmental exposures/habits. The physical exam includes evaluating basic vital signs, body mass index, thyroid evaluation, excess androgen, and pelvic examination.  

For the female evaluation, day 3 follicle-stimulating hormone (FSH), estradiol (E2), antral follicle count, and Anti Mullerian Hormone (AMH) are determined via bloodwork and baseline transvaginal ultrasound to evaluate ovarian reserve. Further medical evaluation in patients with ovulatory dysfunction includes collections of thyroid-stimulating hormone (TSH), prolactin, DHEAS, testosterone, and 17 hydroxy-progesterone.  The above hormonal assessment helps determine the etiology of anovulation. In addition, the uterine and pelvic cavity is also typically evaluated with a baseline transvaginal ultrasound, often in conjunction with a hysterosalpingogram or sonohysterogram. The ultrasound evaluation helps to identify any uterine factors affecting fertility and pregnancy maintenance, such as the presence of polyps, submucosal fibroids, and/or uterine malformations such as uterine septums.

Ultrasound evaluation can also determine other causes of subfertility, such as hydrosalpinx or endometriosis. Male infertility workup includes a semen analysis. Both male and female evaluation usually involves a basic infectious disease workup, including syphilis, a hepatitis panel, and human immunodeficiency virus (HIV).

Technique or Treatment

In vitro fertilization is the most commonly utilized assisted reproductive technology. It involves the collection of oocytes from the ovary, followed by fertilization in vitro, and is completed with transferring the resulting embryo into a uterus. It involves various steps outlined below, including controlled ovarian stimulation, oocyte retrieval, fertilization, embryo culture, and embryo transfer. Additionally, preimplantation genetic testing and intracytoplasmic sperm injection may also be included in the process. Cryopreservation with vitrification is then used to freeze excess embryos or for fertility preservation of eggs or embryos.  

Controlled Ovarian Stimulation

The first cases of IVF utilized a natural menstrual cycle where a single oocyte was retrieved. Natural cycle IVF is still in practice. However, controlled stimulation of the ovaries is now more commonly performed to maximize the number of oocytes gained per cycle. The latter also offers a much higher chance of pregnancy success. 

Multiple agents and regimens exist for controlled ovarian stimulation. Selective estrogen receptor modulators (SERM) like clomiphene citrate and tamoxifen are one such avenue for this.  Benefits of minimal stimulation protocols ("mini-IVF") utilizing SERMs include a decreased risk of ovarian hyperstimulation syndrome and multifetal gestation. However, it also results in a lower live birth rate (49% versus 63% birth rate between mini-IVF and conventional IVF, respectively).[9] 

Injection of exogenous gonadotropins, like follicle-stimulating hormone (FSH) and luteinizing hormone (LH), is frequently used for controlled stimulation. It maximizes the number of developing follicles during a single cycle. The following gonadotropin protocols are most popular currently.  

Gonadotropin-releasing hormone (GnRH) Antagonist cycles- Mixed gonadotropin medications are used through two types of injection, one with FSH activity (Recombinant FSH) and one which has both FSH and LH activity. The premature LH surge is protected from occurring by a GnRH antagonist. These cycles can be started with menses after normal baseline parameters are confirmed with ultrasound and normal hormonal levels of FSH and estradiol levels. On many occasions, the cycle starts after pretreatment with oral contraceptives for 2-4 weeks. 

GnRH Agonist cycles- Mixed gonadotropins are also used through two types of injection, one with FSH activity (Recombinant FSH) and one with both FSH and LH activity. The premature LH surge is protected from occurring by a GnRH agonist. The GnRH agonist is started in the luteal phase of the cycle before the gonadotropins are started. When the gonadotropins are started, the dose of the GnRH agonist is customarily halved until a trigger is given for final maturation before egg retrieval. Pretreatment with oral contraceptives can also be used in these cycles.

Transvaginal ultrasound monitoring is utilized to track the number and growth of follicles. Serum lab testing is also performed, most frequently measuring estradiol (E2) levels to track ovarian response. Once ovarian follicles are mature (typically when 2-3 follicles reach 18mm in size), final maturation is artificially triggered via exogenous human chorionic gonadotropin (hCG) or with a GnRH agonist administration. 

Specific dose-based regimens and other less common stimulation protocols are beyond the scope of this review. 

Oocyte Retrieval 

At its inception, oocyte retrieval was initially performed via laparotomy, then by laparoscopy.[10] It is standardly performed today vaginally using ultrasound guidance under general anesthesia or conscious sedation.[11] A needle is inserted trans-vaginally and guided with ultrasound into each follicle to aspirate the follicular fluid, usually containing an oocyte.  

Greater numbers of oocytes retrieved (up to 15) are associated with improved live birth outcomes.[12] 

Before the ultrasound-guided transvaginal approach, laparoscopic or abdominal retrievals of oocytes were performed. While largely fallen out of practice, laparoscopic and abdominal egg retrievals are occasionally used to obtain oocytes, such as in cases of vaginal agenesis or when the ovaries are not in the pelvis (such as in the case of ovarian transposition in advance of radiation therapy or with patients who have a huge fibroid uterus). 

Fertilization 

Fertilization occurs in vitro by mixing oocytes obtained from retrieval with spermatozoa in a culture medium. Spermatozoa are obtained via an ejaculation sample or surgical retrieval in cases of obstructive azoospermia and isolated via density centrifugation and washing in culture media. For patients with a history of fertilization failure or with male factor infertility, intracytoplasmic sperm injection (ICSI) is considered as it has been shown to improve fertilization rates.[13] ICSI uses a single sperm that is injected directly into the cytoplasm of the oocyte. Embryologists choose sperm for ICSI utilizing morphologic parameters. 

Embryos are incubated for either a day 3 (cleavage stage) or day 5 (blastocyst stage) transfer. Day 5 transfers are more common and have a higher chance of success. Evaluation of the appearance of embryos is more predictive of success on day 5 at the blastocyst stage rather than day 2 or 3 at the cleavage stage in which the embryos are merely 4 or 8 cells, respectively.[14][15]

Preimplantation Genetic Testing (PGT) 

Preimplantation genetic testing is commonly performed in conjunction with IVF. It refers to various genetic assays performed on embryos before transfer to identify possible genetic disorders. For example, preimplantation genetic testing for aneuploidy (PGT-A) screens for whole chromosome abnormalities, whereas preimplantation genetic testing for monogenic disorders (PGT-M) screens for single-gene disorders in high-risk patients. 

PGT can be performed with polar bodies or an isolated blastomere of a cleavage-stage embryo, usually from a day 3 embryo. It can also be performed from a sample of cells from the trophectoderm of a blastocyst-stage embryo. The benefit of a trophectoderm sample is avoiding biopsying the inner-cell mass that gives rise to the fetus. However, in the case of mosaicism, it may not represent the constitution of fetal tissue.[16] There is also the possibility of false-positive results. Diagnostic testing, such as chorionic villous sampling, should be offered during pregnancy. PGT-A was initially performed using fluorescence in situ hybridization (FISH) analysis that utilized selected chromosomes. Most evidence with this method did not show improvement in IVF outcomes.[17] Now, PGT-A is routinely performed on 24 chromosomes, initially with array CGH (aCGH), and now with the use of more advanced technology such as next-generation sequencing.[18] 

With 24 chromosome analysis, evidence for PGT-A is more mixed. A few randomized controlled trials (RCT) comparing elective single-embryo transfer (eSET) of euploid embryos determined by PGT-A versus morphology grade-based selection of embryos found non-inferior or improved pregnancy rates amongst the PGT-A group.[19][20] A 2011-2012 retrospective data analysis from a national ART database found a possible increase in the likelihood of live births in women aged >37 who utilized PGT-A.[21] These current studies are limited either in retrospective design or in the use of patient cohorts with favorable prognoses. Additionally, these studies utilize now infrequently used genetic analysis methods such as aCGH. 

Secondary to the mixed, limited data, ASRM currently does not recommend for or against the universal use of PGT-A. However, it does acknowledge potential benefits, particularly in eSET candidates, and the resultant decrease in multi-gestation pregnancies (discussed below).[22] 

While PGT is classically used for selection against aneuploidies and genetic disorders, it can be used in more ethically controversial avenues such as in sex selection. Additionally, individuals in specific communities, such as the dwarf and deaf community, have been reported to request selection for dwarfism or genetic deafness. A shared decision-making model is recommended in such circumstances.[23][24] 

In Vitro Maturation

In vitro maturation (IVM) is an alteration to traditional IVF, which can be used in select patients, such as those at risk of ovarian hyperstimulation syndrome (i.e., PCOS patients) or women with estrogen-sensitive cancers requiring time-sensitive gonadotoxic treatment. In IVM, immature follicles are collected with minimized to no exposure to hormonal stimulation during the germinal vesicle to metaphase II stage. Typically, a short course of FSH administration is performed, with or without hCG administration for follicular priming.[25] Retrieval and culture need to be modified to obtain and mature the immature oocytes before fertilization. Candidates for IVM are again those that have a relative contraindication to IVF, and patients should be counseled that the rate of achievement to blastocyst stage is lower with IVM. Likely, pregnancy rates are lower with this method as well.[26][27] 

Embryo Transfer

The American Society of Reproductive Medicine (ASRM) developed a standardized protocol for embryo transfer based on a task force that evaluated survey responses regarding individual practices amongst Society for Assisted Reproductive Technology (SART) medical directors.[28] The impetus for developing a standardized practice guideline was based on evidence of limited training in embryo transfer in fellowships and varying IVF outcomes based on the provider performing the transfer. 

The protocol guideline based on the survey and existing evidence is as follows: 

1. One should prepare for the embryo transfer by reviewing prior mock/transfer notes 
2. Patient preparation for the procedure should include analgesics for patient comfort. However, analgesics are not shown to improve pregnancy outcomes. 
3. Checklist-based time out process to ensure appropriate patient and embryo identification 
4. Transabdominal guidance to visualize the endometrial cavity and pelvic anatomy, as well as for ultrasound guidance of the transfer 
5. Standard sterility preparation with hand-washing and sterile gloves 
6. Placement of the speculum. Flushing of the vagina is recommended with either a cotton swab or gauze utilizing saline or media as the cleansing solution 
7. Removal of mucus from the cervical-endocervical canal, with some evidence for improvement in clinical pregnancy rates.[29][30] 
8. Use a soft embryo transfer catheter to pass through the cervix into the endometrial cavity. The transfer may occur directly in which the catheter is loaded with the embryos before catheter placement, with a trial transfer followed by the actual transfer (empty catheter is passed through the cervix before loading the catheter with an embryo for transfer), or the afterload transfer (The catheter is passed through the cervix, after which the inner catheter is removed, with the outer catheter left in place in the canal. The inner catheter is then loaded with the embryo(s) and replaced for the placement of the embryo(s) into the uterus). 
9. One should place the tip of the catheter in the upper or middle third of the endometrial cavity. There is some evidence this position improves pregnancy rates.[31][32] 
10. One should confirm the catheter does not have retained embryo(s) 
11. There is no evidence for bed rest after embryo transfer.[33]   

While the vast majority of embryo transfers are performed in this manner trans-vaginally, for patients with uterine anomalies or difficult trans-cervical access, intrafallopian transfer via gamete intrafallopian transfer (GIFT) or zygote intrafallopian transfer (ZIFT) is a possible alternative laparoscopic transfer technique. 

Current ASRM guidelines recommend single embryo transfer in patients with a favorable prognosis, considering a maximum of two and three blastocyst embryos transferred at once in the 38 to 40 and 41 to 42 age group, respectively.[34] These guidelines are made to reduce multifetal gestation (discussed further in "Complications"). 

Following the embryo transfer, the luteal phase is typically supported with progesterone and estrogen supplementation to promote implantation and pregnancy continuation. 

Cryopreservation 

Embryos not used in the current cycle can be cryopreserved. Cryopreservation is typically achieved with vitrification, a rapid freezing process. Vitrification is thought to prevent cryoinjury by decreasing the development of intracellular crystals.[35] All embryos can also be frozen in patients at risk for ovarian hyperstimulation syndrome (discussed further in "Complications"). The embryos from these "freeze-all" cycles can then be used in a future cycle not associated with the controlled ovarian stimulation. Freeze-all cycles are also utilized in some clinics routinely, as frozen-thawed embryos have been associated with improved pregnancy rates and obstetric outcomes, such as a decreased risk of perinatal mortality and preterm birth.[36][37]

Complications

Ovarian Hyperstimulation Syndrome

Ovarian hyperstimulation syndrome (OHSS) is a potentially fatal complication of controlled ovarian stimulation. The reported incidence of OHSS varies based on diagnostic criteria but is estimated to range between 1 to 5% for moderate/severe cases.[12] 

OHSS develops from the exogenous administration of gonadotropins followed by the administration of hCG. In this process, many follicles recruited and developed in a single cycle result in excessive production of various growth factors such as vascular endothelial growth factor (VEGF) and subsequent neovascularization. The combined excessive follicular growth and fluid and vascular development result in significant fluid shifts to the third compartment, resulting in ascites, edema, pleural effusion, renal injury, pericardial effusion, and thromboembolism. Patients at particular risk of OHSS include those with a diagnosis of PCOS, multi-follicular development, large number of oocytes retrieved (>24), and elevated estradiol levels (>3500 pg/mL).[38][39]

For patients diagnosed with moderate to severe OHSS, treatment involves supportive care with fluid resuscitation, therapeutic paracentesis, and prophylactic anticoagulation. Patients at risk for OHSS must not have a fresh transfer and freeze all embryos until their ovarian stimulation has subsided. The final maturation trigger should be changed from HCG to a GnRH agonist if possible. In addition, cabergoline can be given daily for eight days after retrieval or after trigger to protect the patient from OHSS further. Then, one to two months later, they can safely proceed with a frozen embryo transfer when the ovarian stimulation has resolved.  

Antenatal and Neonatal Complications

The most common complication of ART is the risk of multifetal pregnancies. In 2009, 41.1% of infants conceived via ART were of multifetal pregnancies (compared to 3.5% of infants in the general population).[40] The effort to reduce multifetal gestation via reducing the number of embryos transferred at once has resulted in a significant reduction—by 2017, 73.6% of ART-conceived infants were from singleton pregnancies.[41][34]

Multifetal gestation pregnancies have both maternal and fetal consequences. Pregnancies have a higher likelihood of being complicated by various antenatal conditions, including hyperemesis gravidarum, gestational diabetes, and hypertensive diseases of pregnancy.[42][43] Multifetal gestation pregnancies also have worse fetal and neonatal outcomes than singleton pregnancies, with a significant increase in preterm birth and the associated increase in the risk of stillbirth (fivefold) and neonatal death (sevenfold).[44][45]

IVF providers limit multifetal gestation pregnancies through several avenues, including low-dose stimulation regimens, close hormone and follicle monitoring during stimulated cycles, and limiting the number of embryos transferred per cycle.[46][47] Once a multifetal gestation is diagnosed, appropriate counseling regarding the increased risks in pregnancy should be discussed with the patient, and the option of multifetal reduction, when appropriate, should be offered.[48]

Beyond multifetal gestation, singleton IVF pregnancies are also possibly associated with various increased risks compared to naturally conceived pregnancies. These risks include perinatal mortality, preterm delivery, low birth weight, cesarean section, placenta previa, placental abruption, and preeclampsia. These possible risks should also be discussed when counseling patients, but with caution, as the quality of data is limited by the existing study designs. Standardized tracking of outcomes of ART pregnancies to include these outcomes would improve the quality of evidence for or against these potential risks of IVF-conceived pregnancies.[49][50] There is also limited evidence of a low-level increased risk of birth defects in patients undergoing IVF, particularly with ICSI, though again quality of evidence is low.[51][52] It is reasonable to offer ultrasonographic surveillance during IVF-conceived pregnancies, such as with fetal echocardiography.

Clinical Significance

ART allows individuals and couples to achieve pregnancy in situations that might not otherwise be possible, such as those with infertility, a history of gonadotoxic therapies, or those with deleterious genetic conditions.  

According to the Center for Disease Control, as of 2017, 1.9% of United States-born infants are conceived with ART. In 2017, approximately 200,000 ART cycles with an embryo transfer were performed, with 78,052 live births in that year.[41] As access, rates of delayed childbearing, and insurance coverage for ART increase, these numbers will likely increase. As such, women’s health and reproductive health providers need to have a basic functioning knowledge of indications and appropriate timing for referral to a reproductive endocrinologist and infertility specialist.

Enhancing Healthcare Team Outcomes

As highlighted throughout this review, assisted reproductive technologies require a cohesive interdisciplinary team that ranges from reproductive endocrinology and infertility physicians and nurses to the andrology/embryology team to psychiatric/mental health support. Communication between these teams is vital to the success of ART and enhancing outcomes.