US Pharm. 2024;49(9):HS-12-HS-16.

ABSTRACT: In vitro fertilization (IVF) is an assisted reproductive technology that is designed to aid individuals in conceiving. Given the complexities of reproduction and infertility, IVF involves several steps and a range of medications. For optimal results, the medication protocols are tailored to each patient and require frequent monitoring and adjustments. It is important for patients to understand the potential side effects and risks associated with the medications and procedures involved with IVF. Comprehensive patient education and individualized treatment plans are essential for enhancing the success rates and overall patient experience with IVF. 

Infertility is defined as the inability to become pregnant after unprotected intercourse: 12 months for women aged <35 years and 6 months for women aged ≥35 years.1 The World Health Organization reports that approximately 20% of the global population experiences infertility due to a variety of reasons.2 The prevalence of infertility demonstrates the importance of assisted reproductive technology (ART), which encompasses treatments and procedures associated with the handling of human eggs or embryos to help achieve pregnancy.1 According to the Society for Assisted Reproductive Technology (SART), in 2022, 2.5% of births in the United States resulted from successful ART cycles.3 The most common and effective form of ART is in vitro fertilization (IVF).4 The IVF process can be summarized in five steps: ovarian suppression and stimulation, egg retrieval, fertilization, embryo development, and transfer.1,5

Evaluation and Pretesting

Before beginning IVF, women typically undergo an evaluation, which includes estimating ovarian reserve, evaluating for uterine abnormalities, and sperm testing.

Ovarian reserve is assessed using bloodwork collected during cycle Days 2 to 5 and a transvaginal ultrasound. Basal follicle–stimulating hormone (FSH), estradiol (E2), and anti-Müllerian hormone (AMH) levels are measured in the blood. Elevated FSH is specific, but not sensitive, for diminished ovarian reserve (DOR). E2 assists with interpreting FSH, as a normal FSH level occurring alongside an elevated E2 level may indicate DOR. AMH demonstrates higher sensitivity than FSH and declines earlier than the FSH rise. AMH is commonly used instead of FSH and E2, given these qualities. The transvaginal ultrasound identifies the number of follicles measuring 2 mm to 10 mm in both ovaries, resulting in the antral follicle count (AFC). The lower the AFC, the fewer eggs are available to mature and subsequently retrieve to create embryos. Currently, the most sensitive and reliable markers of ovarian reserve are AMH and AFC (see TABLE 1).6,7

The uterine cavity should be evaluated to rule out the presence of physical abnormalities, such as uterine polyps, fibroids, scar tissue, or septum, which may affect fertility. One of three methods may be used: hysterosalpingogram, hysteroscopy, or a saline infusion sonohysterography.6,7

A semen sample should be collected and analyzed to assess sperm quality, including volume, concentration, morphology, and motility.8 Sperm quality affects overall IVF success and determines which fertilization method is used: standard insemination or intracytoplasmic sperm injection (ICSI). Fertilization is discussed in a subsequent section.6

Lastly, both partners must undergo necessary testing, including HIV, hepatitis B and C, and syphilis. Depending on the patient’s family history and ethnic background, additional tests may be recommended. Once the necessary pretreatment evaluations are complete, the provider will initiate prestimulation treatment.6

Prestimulation Treatment

Estrogen Therapy: An estrogen-containing oral contraceptive or estrogen-only pill may be initiated before starting the ART cycle. Estrogen therapy helps to synchronize irregular menstrual cycles, coordinating ovulation suppression and ovarian stimulation medications. Additionally, evidence suggests that estrogen-containing pills help to prevent the development of ovarian cysts, which could result from gonadotropin-releasing hormone (GnRH) analogues and delay therapy.9-11

Protocols: Various protocols may be used and should be carefully selected by the provider for each patient. This article will focus on the most common protocols. It is important to note that the schedules are estimates; the exact timing of medications may differ among patients due to variability in response. 

The antagonist protocol, often called the short protocol, is the most common, likely due to several advantages (see FIGURE 1). This protocol does not utilize a GnRH agonist for ovulation suppression, which reduces the number of injections for the patient, and it is also the most appropriate for patients who are at increased risk of ovarian hyperstimulation syndrome (OHSS).12,13

The agonist downregulation protocol, also referred to as the long protocol, is another common protocol (see FIGURE 2). This protocol utilizes a GnRH agonist for ovulation suppression and thereby requires more injections compared with other regimens. This protocol tends to produce more mature eggs, though, and is beneficial if premature ovulation is a concern.12,13

The flare protocol is typically reserved for patients who have not responded to other protocols or are of advanced maternal age (see FIGURE 3). While this protocol uses the same medications as the long protocol, they are administered on a different schedule. By administering a GnRH agonist on cycle Day 1, the body will produce a “flare” of endogenous FSH in addition to the exogenous FSH being injected during the stimulation phase, often resulting in an increased number of eggs available for retrieval.12,13

Ovulation Suppression: GnRH agonists or antagonists prevent the luteinizing hormone (LH) surge, which ultimately serves to prevent ovulation from occurring before egg retrieval. Before the use of GnRH agonists, up to 50% of cycles were canceled due to premature ovulation, whereas <5% of cycles are now canceled for this reason.12 In addition, this step helps to increase the number of eggs available for retrieval by synchronizing the eggs to mature at a similar rate during the stimulation phase.14,15 

GnRH Agonist: A GnRH agonist is used in the downregulation (long) and flare protocols. Initially, FSH and LH surge from receptor stimulation, but after 7 to 10 days, receptor desensitization and downregulation of FSH and LH results. Due to their mechanism of action, GnRH agonists must be started before ovarian stimulation to allow time for FSH and LH suppression and subsequent ovulation. Side effects include hot flashes, vaginal dryness, mood swings, headaches, spotting, and injection-site reactions (leuprolide only).12,16

Leuprolide (1 mg/0.2 mL) is the most commonly used GnRH agonist, with a starting dosage of 0.5 mg to 1 mg injected SC once daily. Of note, leuprolide is not FDA approved for use in IVF treatment, though it has been used off-label for decades.12,16 Alternatively, nafarelin, a nasal spray with an initial dosage of two sprays twice daily, may be used. When employed as part of the long protocol, leuprolide or nafarelin should be started approximately 1 week before the expected menstrual cycle. In contrast, for the flare protocol, these agents would be started on menstrual cycle Day 1. The doses of both drugs should be reduced by 50% when ovarian stimulation begins, then continued until the day of the trigger injection.12

GnRH Antagonist: A GnRH antagonist is used in the antagonist (short) protocol owing to its ability to directly and immediately inhibit FSH and LH production. Unlike GnRH agonists, these agents do not cause an initial surge of FSH and LH and do not need to be started before ovarian stimulation. Side effects include hot flashes, vaginal dryness, mood swings, headaches, spotting, and injection-site reactions.

Currently available GnRH antagonists for use in IVF patients are cetrorelix and ganirelix. Cetrorelix (0.25 mg) during the early-to-mid follicular phase and ganirelix (250 mcg) during the mid-to-late follicular phase are both administered SC once daily.12,16 Initiation is based on follicular growth and/or rising estrogen levels, commonly around Day 6 of ovarian stimulation therapy.12

Baseline bloodwork and an ultrasound are typically ordered on cycle Day 3. Specifically, an E2 level <50 pg/mL and the absence of follicles >15 mm indicates ovulation suppression. If either criterion is not met, further evaluation is warranted.17 Once adequate ovarian suppression is achieved, the patient can proceed to the stimulation phase.

Ovarian Stimulation

Compared with one egg typically maturing in a nonmedicated cycle, ovarian stimulation serves to increase the number of mature eggs for retrieval. Ovarian stimulation usually starts on cycle Day 2 or 3 and lasts 7 to 12 days. During the stimulation phase, patients’ follicle development is closely monitored via E2 levels and ultrasounds every 1 to 3 days.5,11-15 It is important to note that the number of eggs available for retrieval is patient- and cycle-dependent. 

Gonadotropins can be used to induce ovulation; currently available agents are menotropins, follitropin alpha, and follitropin beta. Menotropin contains FSH and LH, which are collected and purified from the urine of postmenopausal women. Comparatively, follitropin alpha and beta products contain only recombinant human FSH. These agents directly stimulate multiple follicles to grow, rather than one. The agent, dosage, and frequency are individualized based on the patient’s age, weight, past medical history, AMH level, and previous response to ovarian stimulation (if applicable). If too little gonadotropin is administered, fewer eggs will be available for retrieval. If too much is administered, the risk of OHSS increases.12,15 For optimal outcomes, the lowest effective dose should be used and adjusted throughout the stimulation phase based on follicular development. 

Based on E2 levels and follicle size, the provider will determine when the eggs are ready for final maturation and administer the trigger injection in preparation for retrieval. The trigger injection is typically human chorionic gonadotropin (hCG) or recombinant chorionic gonadotropin. This injection directly activates the LH receptors over 24 to 36 hours to mimic the LH surge prior to ovulation and remains in the body for upward of 8 to 10 days, placing patients at risk of OHSS.5,12

While the development of ovarian cysts is common with these medications, the majority cause little discomfort and resolve spontaneously. Additionally, ovary enlargement can result, demonstrated by abdominal distension and discomfort. Most cases are uncomplicated, are mild-to-moderate in severity, and resolve in 2 to 3 weeks.

In contrast, OHSS is a rare but serious risk associated with hCG injections, leading to ovary enlargement and a fluid shift (e.g., abdominal ascites, pleural effusion). Risk factors for OHSS include history of OHSS, polycystic ovary syndrome, AFC >24, and AMH >3.5 ng/mL. Ovarian stimulation risk factors include >17 follicles that are >10 mm at trigger, E2 >3,500 pg/mL at trigger, and >15 eggs retrieved. The following symptoms may range in severity: abdominal pain and bloating, nausea, vomiting, diarrhea, ovarian tenderness, rapid weight gain (>1 kg in 24 hours), shortness of breath, and decreased urination. Patients who experience any of these symptoms should contact their provider, as moderate-to-severe cases of OHSS require hospitalization for close monitoring and supportive care.5,18,19

After the trigger injection is administered, gonadotropin therapy should be discontinued. Once the eggs undergo final maturation, retrieval will soon follow.

Egg Retrieval

Transvaginal ultrasound aspiration is a procedure to retrieve the egg(s), which takes place under sedation in the provider’s office 34 to 36 hours after the trigger injection was administered. For each egg, an ultrasound guides a thin needle through the vaginal wall and ovary into a follicle to suction the egg. Once all eggs are retrieved, they are placed in a nutrient-dense liquid to help them develop.11,21 On average, 75% of the eggs will mature and be able to move forward with fertilization.14 This 30-minute procedure is generally well tolerated, though patients may experience some cramping and bloating afterward.21 Patients are advised to rest for 24 hours after the procedure.20

Luteal Phase Support (LPS): In preparation for embryo transfer, patients are often started on supplemental progesterone to support the uterine lining and assist in successful implantation. IVF affects the ovaries’ ability to produce adequate progesterone due to GnRH analogues and egg retrieval.22 Multiple regimens are available and administered via IM injection or vaginal route (capsules, gel, or suppositories).16 If the patient becomes pregnant, progesterone therapy is typically continued for the first 8 to 10 weeks of pregnancy until the placenta begins producing progesterone. Additionally, estrogen may be initiated in combination with progesterone for LPS, though the potential benefit remains unclear.22

Fertilization and Embryo Development

Fertilization: Once the mature eggs have been identified, the fertilization process can begin using partner or donor sperm. Two fertilization methods are available: conventional (standard insemination) or ICSI.21 During conventional insemination, the eggs and sperm are placed together in a dish and the sperm are relied upon to penetrate the egg. If the ICSI technique is utilized, an individual sperm is injected directly inside of the mature egg. While this technique was first developed to assist cases of male factor infertility (e.g., low sperm quantity or quality), it may also be used if prior IVF cycles have failed.5,21,23 On average, 70% of the mature eggs will undergo successful fertilization; up to 50% of fertilized eggs will be suitable for transfer.14

Embryo Development: After fertilization, the embryos are closely monitored by an embryologist and grow for upwards of 3 to 5 days. Embryos may be transferred at any time during Days 3 to 5. Patients may elect for an immediate fresh embryo transfer or freeze their embryos for a later frozen embryo transfer (FET).5,11 FET is recommended for patients at risk for OHSS, as evidenced by elevated E2 levels or high ovarian response, to reduce the risk of experiencing moderate-to-severe OHSS.18 If the embryos undergo genetic testing, FET is often required due to the timing and reporting of results.

Assisted Hatching: For successful implantation to occur, the blastocyst must “hatch” from the zona pellucida (outer shell) covering the embryo.23,24 Assisted hatching, done immediately before transfer, is the process of using a laser or acid solution to create a small hole in the zona pellucida. This procedure is not recommended for most IVF patients, though it may be used if previous IVF cycles have failed or if the patient has a poor prognosis of conceiving.24

Genetic Testing: Once an embryo reaches the blastocyst stage (Day 5), patients may opt for preimplantation genetic testing (PGT) to identify genetic defects that may cause implantation failure, miscarriage, or potential birth defects. Three types of PGT may be performed on the embryos: aneuploidy, monogenic disease, and structural rearrangement. Embryos found to have genetic defects are not suitable for transfer.25

Embryo Transfer

Determining the number of embryos to transfer is controversial among providers. An elective single-embryo transfer (eSET) consists of transferring one embryo during a cycle; however, two or more embryos may be transferred to increase the chances of a successful pregnancy. eSET is often recommended for patients aged <35 years with a suitable quantity of quality embryos.5,11 According to 2022 SART data, eSETs accounted for nearly 75% of all transfers.3 Transferring more than one embryo increases the risk of multiple births (e.g., twins) and potential health risks (e.g., preterm labor, fetal loss). Considering this, determining the number of embryos to transfer should ultimately be individualized by accounting for the embryos’ quality and quantity and the patient’s age, past medical history, and IVF history (e.g., failures).5

Embryo transfer is typically performed using a Day 3 cleavage-stage embryo or a Day 5 blastocyst. Cleavage-stage embryo transfers are preferred for patients with fewer quality embryos, while blastocyst transfers are preferred for patients who have failed a Day 3 transfer or have many quality embryos.5 During the transfer procedure, a provider places the fresh or frozen embryo(s) directly into the uterus using an ultrasound-guided catheter. This procedure takes a few minutes, does not require sedation, and is generally painless. After the embryo transfer, patients may experience mild bloating, cramping, and spotting.11,21

Follow-Up: A pregnancy test should be completed 9 to 13 days after the embryo transfer. To reduce the likelihood of a false-positive result, patients who underwent a fresh embryo transfer with an hCG trigger injection should avoid testing too early (<14 days from the injection).15 If the test returns positive, the patient will be closely monitored to ensure that the pregnancy is progressing normally. If the test returns negative, the patient should follow up with her provider to discuss next steps.

Conclusion and Pharmacist’s Role

IVF treatment is complex, requires multiple agents, and is tailored to each patient and her response. Most medications used during IVF treatment are dispensed from a specialty pharmacy and sent directly to the patient’s home. Although most community pharmacists will not distribute these medications, they are the most accessible healthcare provider and are positioned to address patients’ questions. It is important, therefore, to be aware of reputable IVF resources, notably the American Society for Reproductive Medicine and its affiliate organization, the Society for Assisted Reproductive Technology.

REFERENCES

1. CDC. Assisted Reproductive Technology (ART) Glossary Terms. December 28, 2023. www.cdc.gov/art/reports/2021/glossary.html. Accessed June 24, 2024.
2. World Health Organization. Infertility prevalence estimates, 1990-2021. April 3, 2023. https://iris.who.int/bitstream/handle/10665/366700/9789240068315-eng.pdf?sequence=1. Accessed June 24, 2024.
3. American Society for Reproductive Medicine. IVF-assisted pregnancies constitute 2.5% of all births in 2022. www.asrm.org/news-and-events/asrm-news/press-releasesbulletins/ivf-assisted-pregnancies-constitute/
#:~:text=In%202022%2C%20the%20number%20of,result%20of%20successful%20ART%20cycles. Published April 16, 2024. Accessed June 24, 2024.
4. Dept. of Health and Human Services. Fact sheet: in vitro fertilization (IVF) use across the United States. March 13, 2024. www.hhs.gov/about/news/2024/03/13/fact-sheet-in-vitro-fertilization-ivf-use-across-united-states.html#:~:text=In%202021%2C%2086%2C146%20infants%20born,children%20at%20a%20later%20time. Accessed June 24, 2024.
5. Women and Infants Fertility Center. In vitro fertilization (IVF). https://fertility.womenandinfants.org/treatment/ivf. Accessed June 24, 2024.
6. Society for Reproductive Technology. Evaluation before IVF. www.sart.org/patients/sart-patient-evaluation/. Accessed June 24, 2024.
7. Practice Committee of the American Society for Reproductive Medicine. Testing and interpreting measures of ovarian reserve: a committee opinion. Fertil Steril. 2020;114(6):1151-1157.
8. UCSF Center for Reproductive Health. Semen analysis. https://crh.ucsf.edu/about-fertility/diagnostic-fertility-testing/semen-analysis. Accessed June 24, 2024.
9. Society for Reproductive Technology. ART: Step-by-Step Guide. www.sart.org/patients/a-patients-guide-to-assisted-reproductive-technology/general-information/art-step-by-step-guide/. Accessed June 24, 2024.
10. Biljan M, Mahutte N, Dean N, et al. Pretreatment with an oral contraceptive is effective in reducing the incidence of functional ovarian cyst formation during pituitary suppression by gonadotropin-releasing hormone analogues. J Assist Reprod Genet. 1998;15(10):599-604.
11. Cleveland Clinic. IVF (in vitro fertilization).  March 2, 2022. https://my.clevelandclinic.org/health/treatments/22457-ivf?utm_medium=social&utm_source=youtube&utm_campaign=cc+yt. Accessed June 24, 2024.
12. Society for Reproductive Technology. ART medications. www.sart.org/patients/a-patients-guide-to-assisted-reproductive-technology/general-information/art-medications/. Accessed June 24, 2024.
13. Alife Health. IVF ovarian stimulation protocols. www.alifehealth.com/blog/ivf-ovarian-stimulation-protocols/. Accessed June 24, 2024.
14. Northwestern Medicine. In vitro fertilization (IVF). https://fertility.nm.org/in-vitro-fertilization.html. Accessed June 24, 2024.
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16. Lexidrugs. UpToDate Lexidrug. UpToDate Inc. https://online.lexi.com. Accessed June 24, 2024.
17. Dallas-Fort Worth Fertility Associates. Injectable Fertility Medications. www.dallasfertility.com/ivf-medication.html. Accessed June 24, 2024.
18. Practice Committee of the American Society for Reproductive Medicine. Prevention of moderate and severe ovarian hyperstimulation syndrome: a guideline. Fertil Steril. 2024;121(2):230-245.
19. Society for Reproductive Technology. Ovarian hyperstimulation syndrome. www.sart.org patients/a-patients-guide-to-assisted-reproductive-technology/stimulation/ovarian-hyperstimulation-syndrome/.
Accessed June 24, 2024.
20. Society for Reproductive Technology. Oocyte retrieval and embryo transfer. www.sart.org/patients/a-patients-guide-to-assisted-
reproductive-technology/general-information/oocyte-retrieval-and-embryo-transfer/. Accessed June 24, 2024.
21. Mayo Clinic. In vitro fertilization (IVF). September 1, 2023. www.mayoclinic.org/tests-procedures/in-vitro-fertilization/about/pac-20384716. Accessed June 24, 2024.
22. American Society for Reproductive Medicine. Progesterone supplementation during IVF. 2016. www.reproductivefacts.org/globalassets/_rf/news-and-publications/bookletsfact-sheets/english-pdf/progesterone_supplementation_during_ivf_factsheet.pdf. Accessed June 24, 2024.
23. Society for Reproductive Technology. Micromanipulation. www.sart.org/patients/a-patients-guide-to-assisted-reproductive-technology/general-information/micromanipulation/. Accessed June 24, 2024.
24. American Society for Reproductive Medicine. Assisted hatching. 2023. www.reproductivefacts.org/globalassets/_rf/news-and-publications/bookletsfact-sheets/english-pdf/assisted_hatching.pdf. Accessed June 24, 2024.
25. Women and Infants Fertility Center. Preimplantation genetic testing (PGT). https://fertility.womenandinfants.org/treatment/preimplantation-genetic-testing. Accessed June 24, 2024.

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