In Vitro Fertilization (IVF)

In vitro fertilization (IVF) is a series of procedures intended to help with fertility or to prevent genetic problems while assisting with the conception of a child. The process involves the collection of mature eggs from ovaries, which are fertilized with collected sperm in a lab. The fertilized egg or eggs are then transferred to a uterus in order to promote a regular pregnancy.

Often a full cycle of IVF takes around three weeks. IVF is one of the most effective forms of reproductive technology, with the procedure capable of being done with a couple's own sperm and eggs, or else involving eggs, sperm, embryos, or all of the above from either known or anonymous donors. In some cases, a gestational carrier, or a surrogate uterus, might be used.

IVF is never the first step in the treatment of fertility, as there are simpler processes of artificial insemination, such as fertility drugs or surgery. However, in the case of complete tubal blockage, IVF can be the first choice; when other artificial insemination methods have failed, IVF is considered. IVF has also been used as a treatment for genetic problems, in which a couple may choose to try and not pass them down to their offspring. The procedure is also offered as a primary treatment for infertility in women over the age of forty. Health conditions IVF can be used for include:

• Fallopian tube damage or blockage
• Ovulation disorders, such as infrequent or absent ovulation
• Endometriosis
• Uterine fibroids, which are benign tumors in the uterus
• Previous tubal sterilization or removal
• Impaired sperm production or function
• Unexplained infertility
• Fertility preservation for cancer or other health conditions in which treatment can threaten fertility
• Genetic disorders, especially in cases where a partner is at risk of passing on this disorder to a child

The risks associated with the in vitro fertilization process include:

• Multiple births—the transfer of more than one embryo is often done to ensure pregnancy, but can result in multiple births
• Premature delivery and low birth weight—evidence suggests IVF increases both risks
• Ovarian hyperstimulation disorder—the use of injectable fertility drugs, such as human chorionic gonadotropin to induce ovulation, can cause ovarian hyperstimulation syndrome in which a woman's ovaries become swollen and painful
• Miscarriage—the rate of miscarriage for a woman conceiving through IVF increases with the age of the mother
• Egg-retrieval procedure complications—the use of an aspirating needle can cause bleeding, infection, or damage to the bowel, bladder, or blood vessel
• Ectopic pregnancy—about 2 to 5 percent of women using IVF have an ectopic pregnancy, or a pregnancy outside of the uterus, often in a fallopian tube
• Birth defects—there is an observed increase in the rate of birth defects in IVF-assisted births, although there is no clear evidence if IVF conception causes an increased risk of birth defects
• Cancer—some studies suggesting a link between medications used to stimulate egg growth and the development of ovarian tumors
• Stress—IVF can lead to financial, physical, and emotional stress

The process of IVF, often called a cycle, starts with preparation, which includes ovarian reserve testing, which involves taking a blood sample for the level of follicle stimulating hormone (FSH). This informs a physician on the size and quality of a woman's eggs. The uterus is also examined, often using an ultrasound or the insertion of a scope, to determine the health of the uterus and the best way to implant the embryos.

Men also go through sperm testing to analyze the number, size, and shape of the sperm, and to check to see if the sperm are weak or damaged—in which case, a procedure called intracytoplasmic sperm injection (ICSI) can be necessary. This involves injecting sperm directly into an egg.

Sometimes, during the preparation for an IVF cycle, the cycle will be canceled before egg retrieval because either there are an inadequate number of follicles developing, premature ovulation, too many follicles developing (which creates a risk of ovarian hyperstimulation syndrome), or other medical issues that are discovered during the preparatory stage.

Following the preparation, and in the case the IVF cycle is approved, there are five steps that compose a full cycle of IVF:

• Stimulation
• Egg retrieval
• Insemination
• Embryo Culture
• Transfer

The first part of the process involves fertility drugs that begin the stimulation process, sometimes call superovulation. The drugs contain follicle stimulating hormones and work to induce the body to produce more than the normal single egg per month. With more eggs, the chances of a successful fertilization later in the process is increased. This process involves regular transvaginal ultrasounds and blood tests to monitor hormone levels and the ovaries.

Before the eggs are scheduled to be retrieved from the body, the patient receives a hormone injection to help the eggs mature quickly. Then there is a minor surgical procedure, called follicular aspiration, to remove the eggs. This is often done as outpatient surgery in a doctor's office. The procedure involves the use of ultrasound to guide a thin needle into each of the ovaries through the vagina. The needle has a device used to suction the eggs out. The procedure is done with the use of medication to help patients deal with any discomfort, although often the patient experiences some cramping post procedure.

Sperm are then collected either from a partner or from a donor. Once collected, the sperm are put through a high-speed wash and spin cycle in order to find the healthiest sperm. These sperm are then used for the insemination. This process can take a few hours for the sperm to fertilize the egg, and sometimes the procedure is done through the direct injection of the sperm into the egg—a process known as intracytoplasmic sperm injection (ICSI).

During this stage, the doctor monitors the fertilized eggs to ensure that they are dividing and developing. This stage can involve testing the embryos for any genetic conditions. This stage often takes three to five days after fertilization occurs.

Following the collection of the eggs, a patient receives further medication intended to help prepare the lining of the uterus to receive the embryos. Often multiple fertilized embryos are transferred back to the patient in order to ensure at least one will implant itself in the lining of the uterus and begin to develop. This can lead to multiple pregnancies. Once the embryo is implanted, the process replicates natural reproduction. The next step in the overall procedure is a pregnancy test to ensure the procedure worked.

The success of in vitro fertilization procedures can depend on a number of factors. These can include the reason for the initial infertility, where the procedure is done, whether the eggs are frozen or fresh, whether the eggs are donated or a patient's own, and the patient's age. The CDC compiles statistics for all assisted reproductive technology procedures performed in the United States, of which IVF accounts for around 99 percent of the procedures. The CDC report from 2018 showed that 50 percent of IVF procedures in women aged 35 and under resulted in live birth. For women ages 42 and older, 3.9 percent of the egg transfers resulted in a birth.

In the case of older patients, specifically those with eggs retrieved after the age of 37, embryos can go through assisted hatching. This involves helping an embryo in the blastocyst stage hatch out of the shell, or the zona pellucida, in order to implant in the uterus. In some situations, the embryo is considered unable or is observed to be unable to properly hatch out of this shell and implant. In these cases, a small laser is used to either thin the shell or make a small hole in the shell. This procedure can also be used to assist patients with poor quality embryos, with previous repeated IVF implantation failures, and for frozen embryos. Assisted hatching may not benefit all individuals undergoing IVF treatment, as there is a theoretical risk of damaging the embryo during assisted hatching.

The IVF procedure is not a simple procedure, and as such can be a costly procedure to undergo. According to the National Infertility Association, the fees for a single cycle of in vitro fertilization, including the medications, procedures, anesthesia, ultrasounds, blood tests, lab work, and embryo storage can be up to $15,000.

Meanwhile, the National Conference of State Legislatures has the average cost of an IVF cycle in the United States costing anywhere from $12,000 to $17,000. They note the price can vary by location, the amount of medications required, and the amount an insurance company will pay towards the procedure. The coverage by insurance companies can vary by state, with some states requiring coverage and others not. As well, some carriers pay for infertility drugs and monitoring, but not the cost of IVF or artificial reproductive technology.

Embryo selection is often based on the embryo morphology and the rate of embryo development in a culture. Positive selection criteria includes the number of blastomeres, the absence of multinucleation, early cleavage to the two-cell stage, and a low percentage of cell fragments in embryos. This process also involves selecting for factors found to increase pregnancy and implantation rates. A sequential assessment model with algorithms is most often used to take factors into account and has been able to select for embryo development into blastocysts in 86 percent of cases.

Research has been conducted on alternative selection methods driven by the concept that embryo selection is essential to optimize the success rates of IVF. This is especially based on the belief that better selection methods should result in higher live birth rates without an increase in multiple pregnancies.

To achieve the best possible live birth rate after IVF, while minimizing the risk for a multiple pregnancy, one or two embryos that are considered to have the best chance are selected for transfer. Supernumerary embryos with a good chance of implanting can also be selected for cryopreservation and possible transfer in the future while any remaining embryos are discarded. One of the methods for embryo selection is morphological evaluation, which involves the evaluation of an embryo for multiple morphological characteristics at one or several stages of preimplantation development, and is part of the selection of embryos for transfer. However, with embryo selection based on morphological evaluation, implantation rates in general do not exceed 35 percent success rates, although varying results have been reported.

A studied alternative to morphological evaluation is preimplantation genetic screening (PGS). The classical form of PGS involves a biopsy at day three of embryo development of a single cell of each of the embryos available in an IVF cycle and the analysis of this cell by fluorescence in-situ hybridization (FISH) for aneuploidies, for a limited number of chromosomes. Only those embryos for which the analyzed blastomere is euploid for the chromosomes tested are transferred. And while PGS has been increasingly used, trials have shown it can decrease ongoing pregnancy rates compared to IVF with morphological selection for embryos.

In an effort to overcome some of the drawbacks of PGS using cleavage stage biopsy and FISH, new methods to determine the ploidy status of a single cell have been developed. This includes comparative genomic hybridization arrays or single nucleotide polymorphism arrays. And increasing time and money have been invested into the development of non-invasive methods to select the best embryos for transfer. These non-invasive methods include metabolomic profiling, amino acid profiling, respiration-rate measurement, and birefringence imaging.

Embryo selection allows for couples at risk of transmitting genetic diseases to ensure their potential future children are unaffected by the disease. This is done through a process known as preimplantation genetic diagnosis, often referred to as embryo screening, which involves extracting a single cell from an eight-cell embryo and analyzing the cell's DNA for the presence of one or more disease-associated genetic alterations. This process can ensure that future children are unaffected by a given genetic disease without needing to go through the process of prenatal diagnosis and without being forced to make a difficult decision regarding pregnancy termination.

PGD was introduced into clinical care in the early 1990s to determine the sex of embryos and minimize the likelihood of transmitting fatal sex-linked disease genes to offspring. However, since the 1990s, the procedure has expanded from embryo sexing to single-gene diagnostic testing. And reproductive clinicians regularly use PGD to diagnose around 170 different conditions. A third, and somewhat controversial use, of PGD involves screening chromosomally abnormal embryos in an effort to improve the relatively low pregnancy rates and decrease the relatively high miscarriage rates associated with in vitro fertilization procedures.

There is consistent research and development in the in vitro fertilization procedure in order to reduce the stress on patients and increase the chance for successful pregnancies. Some of these include:

• Three-parent procreation: researchers used a technique called mitochondrial replacement therapy (MRT) in IVF, which led to the birth of a baby boy. MRT uses several methods that combine the DNA of one male and one female partner with the mitochondrial DNA of a female donor with the aim of enabling women with rare genetic diseases to avoid passing these conditions on to their children.
• Drugs to reduce the risk of ovarian hyperstimulation syndrome (OHSS), which is rare in its severest form but appears often in as many as one-third of IVF cases, according to the Royal College of Obstetricians and Gynaecologists. The drug in development is a kisspeptin agonist, which stimulates the hormonal pathway responsible for the onset of puberty and can trigger ovulation.
• Guidelines by the Japanese Society of Obstetrics and Gynecology (JSOG) have been developed for the use of preimplantation genetic testing (PGT) by limiting it to JSOG-approved clinics that can perform the technique as part of the research. Patients are also vetted and subject to approval by an ethics committee, which has reduced the rate PGT is used in Japan.

With the increase in machine learning and artificial intelligence, there has been the development of PGTai technology, which uses AI and ML to improve preimplantation genetic testing. The technology uses the test results from biopsy samples taken from embryos that resulted in healthy life at birth to determine which embryos have the correct number of chromosomes. The use of AI and algorithms in the process makes the system more robust and less prone to the subjective calls of a physician undertaking their own analysis.

Another growing area of research and development for IVF is helping women get pregnant. Although the rate of improvement in IVF was rapid at first, the improvement has since slowed, and the developments, additions, and marginal gains made to products, processes, and procedures are therefore key. Another area of focus is the improvement of implantation rates and the reduction of miscarriage rates.

As well, a multinational research team used artificial intelligence to differentiate between poor-quality and high-quality early-stage human embryos. This research project was intended to help reduce the bias in the embryo selection process and overall improve clinical outcomes. The study found that AI can determine more accurately than a human clinician whether an embryo stands a greater likelihood of progressing to a successful implantation and result in a successful pregnancy.

The use of digital technology in reproductive science has traditionally involved investigation into determining and maintaining basic laboratory conditions. More recently, this technology has been used in precision research and evidence-based clinical advances to ensure optimal embryo development and selection and improved outcomes for patients. While in some cases the IVF procedure and fundamentals are static, novel technologies offer the promise of incremental gains, which have become important for improved clinical outcomes for patients.

One of these incremental improvements is the use of automated and digital technology to help maintain the internal temperature and gas concentrations for the environment for gametes and embryos to thrive. This internal environment can be continually monitored and any deviation from the ideal can trigger both audible and external alarms. This offers a chance to develop a consistent and optimal environment for the embryos.

Also known as IVF-on-a-chip, microfluidics and IVF have a lot in common, with both driving the evolution of concepts, designs, methodology, and applications in either field. Many new microfluidic approaches have been proposed and investigated for IVF procedures and to serve unmet needs for automation. For example, microfluidics have been used to improve the selection of sperm, facilitating the processing of oocytes, growing ovarian follicles, and incubating the embryo.

Sperm selection can be challenging, with traditional sperm selection methods such as swim-up and density gradient centrifugation being time and labor intensive and yielding low amounts of motile sperm or high DNA fragmentation. To address these challenges, microfluidics have been integrated with procedures such as semen analysis for male infertility diagnosis and sperm selection, which is based on motility and facilitated by chemotaxis, rheotaxis, thermotaxis, or inertial focusing.

One such device was developed using a polycarbonate membrane filter and gravity to sort for the most motile and functional sperm, which would have to swim against gravity and through the filter into a retrieval reservoir at the top of the filter, leaving dead and less motile sperm behind. This device, in research, was able to sort unprocessed semen samples with higher DNA integrity and fewer reactive oxygen species than traditional sperm-sorting methods.

Another device selected sperm based on the progressive motility in 500 radial microchannels. This device required one step to purify and select the sperm of high DNA integrity from 1 mL of raw semen within less than twenty minutes. Compared to traditional practices of either swim-up or centrifugation, these experiments, done with bull sperm, indicated more than an 89 percent improvement in the selected sperms' motility, and clinical tests using human sperm showed a more than 80 percent improvement in DNA integrity.

In addition, another research group used the principle of thermotaxis to evaluate human sperm on a microfluidic device through an interfacial valve. The temperature gradient was established and controlled by an external system for the greatest possible accuracy. The test showed that human-responsive sperm have a tendency to accumulate in the higher-temperature area in the four tested temperature ranges. The device then trapped these sperm in a discrete branch by an air-liquid interfacial valve.

Another microfluidic device for sperm selection, named the Simple Periodic ARray for Trapping And IsolatioN (SPARTAN), was developed using an array of pillars to isolate motile and morphologically normal sperm from raw semen with lower epigenetic global methylation. This device modulated the directional persistence of sperm in order to increase the separation between progressive and non-progressive motile sperm populations within ten minutes, compared to the sixty to ninety minutes with the traditional swim-up technique. The proposed microfluidic method yielded significantly reduced DNA fragmentation, improved morphology, and nuclear maturity.

Microfluidics have also been demonstrated to be a powerful tool to understand how sperm compete for the insemination of the oocyte in vivo. This has shown that sperm with the highest motility are the most likely to make it through the narrow junctions inside the female reproductive tract, by using an hourglass shaped microfluidic device to simulate the fluid mechanical properties of these narrow junctions in vitro. These oocytes are natively surrounded by cumulus cells, which form an organized structure known as the cumulus-oocyte complex (COC). In vivo, the cumulus cell mass is naturally removed from the oocyte upon fertilization. However, in IVF clinics, oocytes are usually denuded from the cumulus cell through a combination of enzymatic action of hyaluronidase and mechanical pipetting. These procedures can be inefficient and suffer from variations between operators. Microfluidics have been used to help automate the denudation procedure and offers the potential to reduce time and cost while improving standardization.

A microfluidic device has been developed to mechanically remove the cumulus cell mass from single bovine zygotes. The zygote is the first stage of the early embryo, following the fusion of the haploid male and female pronucleus. In the proposed device, the cumulus cell mass was partially removed and reoriented after the cumulus-zygote complex passed through a constricted channel. Two suction points, significantly narrower than the complex, were used to remove the remaining cumulus cell mass. The device processed one zygote at a time and required manual controlling and switching of multiple fluid flows to achieve positioning and movement of the cumulus-zygote complex.

Another device was developed that denuded mouse oocytes from the surrounding cumulus cell mass for ICSI. This device put hyaluronidase-treated COCs at either the germinal-vesicle or metaphase II stage passed through a series of jagged-surface constriction microchannels of optimized geometries. Denudation efficiency of more than 90 percent was achieved when processing COCs through at least 100 repeats. Oocytes that were denuded by the device showed comparable fertilization and developmental competence compared with traditional mechanical pipetting technique. In addition, computational simulations in this study revealed that on-chip denudation was able to generate a smaller shear than manual denudation and imposed less mechanical stress on the cells.

A separate device was developed to celebrate high-quality bovine oocytes to improve the conception rate of IVF. The device featured a long separation channel. After denuded oocytes were injected into the separation channel, they traveled towards the outlet at an optimal flow rate controlled by a syringe pump and settled gradually. The outlets of the separation channel were divided into upper and lower reservoirs. High-quality oocytes settled faster than poor-quality ones in a phosphate-buffered saline buffer. This allowed for the collection of high-quality oocytes and IVF results showed that the development rate of blastocysts of oocytes collected from the lower outlet reservoir were significantly higher than those collected from the upper outlet.

The in vitro culturing of ovarian follicles provides the possibility of restoring fertility for patients facing premature infertility, especially in the case of infertility due to cancer therapies. One such developed device was a three-dimensional culture system to support in vitro development of follicles, producing mature oocytes capable of fertilization. The device used immature follicles encapsulated within sterile alginate beads, which were able to maintain the oocytes' three-dimensional architecture and cell-cell interactions.

Another system was developed to support ovarian follicles in mice and to produce the human 28-d menstrual cycle hormone profile, which controlled human female reproductive tract and peripheral tissue dynamics in single, dual, or multiple unit microfluidic platforms. The mouse ovarian tissue was cultured on a microfluidic platform based on pneumatic actuation, which simulated the in vivo female reproductive tract, and the endocrine loops between organ modules. These included ovary, fallopian tube, uterus, cervix, and liver. This platform achieved organ-organ integration of hormonal signaling and pregnancy-like endocrine loops and presented a possible tool for drug discovery and toxicology research.

An integrated microfluidic system is expected to be able to achieve embryo incubation while reducing overall human intervention in order to increase efficiencies and reduce operator-based variation. This has seen in vitro insemination, co-incubation, and fertilization of murine oocytes in a microfluidic chamber being demonstrated. The device used a barrier gate that allowed only media and sperm to flow through, while the oocyte was blocked without deformation. This selective passage resulting in increasing the local sperm concentration around the trapped oocyte, and the interaction was increased compared with microdrop-in-a-dish technique and demonstrated that a smaller total number and concentration sperm could achieve similar fertilization rates to corresponding techniques.

A similar system was developed featuring microwells that facilitated single oocyte trapping, fertilization, and embryo culture. This device offered a comparable fertilization rate to the traditional microdrop-in-a-dish technique, with the potential to simplify oocyte handling and manipulation, for rapid and convenient medium changing and offering automated tracking of any single-embryo development. Where another study engineered a microfluidic device with hydrodynamic traps to longitudinally monitor viable, separate single oocytes, including perfusion of stimulation media and the introduction of sperm for insemination and prolonged incubation at controlled temperatures.

A different microfluidic device was developed to integrate each step of IVF, including oocyte positioning, sperm screening, fertilization, medium replacement, and embryo culture. The individual oocytes were positioned in an array of octa-column units. The array was connected to four channels, which formed a cross with the oocyte positioning array in the center, allowing motile sperm selection and facilitating rapid medium replacement. By passing through the screening channels, the average mouse sperm used in the development of the device was increased from an average of 60.8 percent to 96.1 percent. Embryo growth rate and blastocyst formation were similar between the microfluidics group and the conventional control group.

Prior to 1978, women without functioning fallopian tubes were largely considered by their physicians. At least one fallopian tube is necessary for the natural fertilization of an oocyte by sperm in vivo. In the past, many women with damaged tubes resorted to reparative surgery or tuboplasty in hopes of re-establishing a conduit for gametes to transfer. These surgeries often failed, until the late 1970s, in which Lesley Brown became the first embryo transfer that resulted in a live birth—the first from IVF.

Initial studies of IVF conducted in women undergoing natural menstrual cycles yielded on average 0.7 oocytes per retrieval and a 6 percent per cycle pregnancy rate. In the 1980s, researchers at the Jones Institute in Norfolk, Virginia, began injecting women with gonadotropins to stimulate multiple ovarian follicles to produce oocytes. The oocytes were then fertilized in vitro, and the healthiest appearing embryos were implanted in the uterus. This advent of controlled ovarian stimulation (COS) improved the average oocyte yields and the average pregnancy rates. These rates saw a rise from 23.5 percent per cycle in 1982 to 30 percent in 1983.

This process initially used human chorionic gonadotropin (hCG) to trigger ovulation and increase the natural cycles. In early IVF procedures, an important concern was premature ovulation, which made retrieving oocytes impossible despite careful and labor-intensive. Two innovations into IVF practice, including the use of gonadotropin releasing hormone (GnRH) agonists in the 1980s and of GnRH antagonists in 2001, making it possible to prevent premature ovulation and control oocyte retrieval.

Since the early days of in vitro embryo culture there have been efforts to improve the culture system to optimize embryo development and increase the number of high-quality embryos available for transfer. Initially, embryo culture media was fashioned from media intended for somatic cells further supplemented with serum. This has since been optimized for embryo metabolism and development by supplementing the media with various macromolecules, altering the energy substrate composition and amino acid balance and added growth factors. Laboratories for many years manufactured their own culture media before these were commercially produced, which resulted in improved consistency and quality control amongst laboratories and practices.

Improvements in embryo culture over the years have allowed in vitro to extend the culture of embryos to the blastocyst stage, permitting detailed morphologic assessment of embryos and better selection of embryos for transfer. These developments have been important for maximizing pregnancy rates in IVF, while minimizing the number of embryos transferred and reducing the overall risk of multiple pregnancies. Extended culture has also allowed for preimplantation genetic testing of embryos, which has been applied to sustain several cells for genetic testing. Improving embryo culture, in combination with other improvements, have allowed for the generation of more embryos than are initially transferred. This has also helped women facing gonadotoxic treatments, such as chemotherapy, to preserve future fertility and have their oocytes received and frozen.

As well, since the 1990s, the options to prevent transmissions of genetic defects have been expanded beyond just invasive techniques, such as chorionic villus sampling and amniocentesis, after which termination could be offered if the fetus was found to be affected. Throughout the 1990s, as surplus embryos became available, techniques were developed to utilize the time between oocyte fertilization as an opportunity to identify which embryos were affected by chromosomal imbalance or a specific gene disorder before transfer to the uterus. This led to cells being biopsied from the trophectoderm of blastocyst-stage embryos where testing can determine whether the embryo carries a mutation or to ensure this trait has not been passed on.