“Assisted reproduction” is the support provided to address infertility issues. It refers to all methods that assist infertile couples to achieve pregnancy and give birth to their own babies. Infertility rate in Pakistan is documented to be 21.9%, and the success rate is only 25% even after introduction of procedures like intracytoplasmic sperm injection (ICSI). “Assisted reproductive techniques (ARTs)” are procedures that represent an amalgamation of development in physiology, endocrinology, pharmacology, diagnostic technology, and clinical care of infertile couples. These techniques are intended to overcome natural barriers in fertilization and include in vitro fertilization (IVF) of human gametes and embryo replacement into the uterine cavity with the goal of achieving pregnancy.
Need of assisted reproductive technology
Since the first successful term birth with the help of ART procedure, continued technological advancements have been made to refine ARTs for improved outcomes. The need for ARTs is correspondingly increasing with increasing awareness, advancement of new techniques, increase in the number and accessibility to fertility clinics, enthusiasm, and affordability of the couples to pursue treatments.
Physiology of reproduction
During the process of reproduction, the male yields millions of gametes or spermatozoa, while the female yields a single female gamete or oocyte. In gametogenesis, haploid gametes are formed by meiosis of diploid progenitor cells that can fuse at the time of fertilization to produce a new, totally exclusive diploid organism. The homologous genetic recombination in prophase of meiosis I generates genetic diversity. This random combination of genetic material produces a variety of traits in the members of the species.
The ejaculation of sperms into the female reproductive tract enables the process of fertilization. While passing through the female reproductive tract, the process of capacitation occurs in sperms with the removal of cholesterol from the membranes along with calcium influx, thus making the plasma membrane less stable. In addition to this, the hyperactivated sperm motility and hyaluronidase secretion helps the sperm to penetrate the cumulus extracellular matrix for reaching the zona pellucida, where the capacitated sperm initiates acrosome reaction by binding of zona protein ZP3 with a sperm protein 1,4-galactosidase. This reaction releases hydrolytic enzymes, including proacrosin, which are activated to acrosin. Acrosin along with the flagellar beating of sperm is important in digesting zona pellucida and penetration of sperm into the oocyte plasma membrane. Then, the plasma membranes of sperm and oocyte fuse, by the interaction of sperm protein; Izumo1, and its cognate receptor, Juno, on the oolemma. Ultimately, sperm nucleus moves into the egg cytoplasm and chromatids of sperm and oocyte condensate to form pronuclei, thus completing meiosis. Polyspermy is blocked by certain changes that include shedding of Juno receptors from the oolemma, oocyte membrane depolarization, and cortical granule secretion.
Diploid zygote is formed by the combination and intermingling of the maternal and paternal chromosomes at syngamy, following the breakdown of pronuclear membranes. After the first cleavage division, two-cell embryo is formed 28 h after insemination, and the embryo undergoes several cleavage divisions forming a four-cell embryo at 44 h and an eight-cell embryo at 68 h. Beyond this eight-cell stage, embryo undergoes cell differentiation and enters the uterine cavity late on day 3 after fertilization. The embryo reaches the blastocyst stage in 5–6 days of fertilization, and after hatching from the zona pellucida, syncytial trophoblastic cells interact directly with the uterine epithelium through the stages of apposition, stable adhesion, and invasion.
Factors affecting successful outcome of ARTs
The physiognomies and lifestyles of the individual undergoing that treatment have an indirect effect on the successful outcome of every procedure. Factors in both partners influence the success of an ART, though some elements have a more significant effect than others.
Maternal age: Female’s age is an important factor to determine the successful outcome of the procedure. The comparative decrease in fertility of women in their late 30s has been reported to be half than the women in early 20s. With a delay in the referral of females to infertility clinics, increased maternal age may contribute to a reduction in maturity and fertilization of oocytes. Increasing age causes a diminution in the reproduction potential of women due to a decrease in the number of follicles, retrieved oocytes, and number of embryos. It is reported that the higher follicle-stimulating hormone (FSH) levels in older patients are responsible for lower implantation rate. Regarding all infertility treatment procedures, chances of success are diminished with age and a corresponding live birth rate is also decreased by 2% for each year with an increase in female age. The live birth rate per IVF cycle is about 26%–40% in females under the age of 35 years and decreases to about 6% above 40 years of age.
Ovarian reserve: The ovarian reserve is mainly tested by follicle stimulating hormone (FSH), estradiol, anti-Müllerian hormone (AMH), and an ovarian antral follicle count (AFC). FSH is routinely done in clinical practice since it can also predict the response to ovarian stimulation.
Body mass index (BMI): Decline in natural fertility has been reported to be associated with obesity (BMI: 25.8–30.8 kg/m 2 ). This can be due to either disturbed hypothalamic–pituitary–ovarian axis or ovulatory dysfunction induced by increased BMI. The rate of infertility in obese women is expected to increase by 4% per kg/m 2 rise in BMI. The success of fertility treatments in obese individuals decreases to half when compared with normal weight individuals. Lower pregnancy rates and spontaneous abortion have also been reported in obese women after IVF or ICSI.
Obesity in men (BMI ≥ 35 kg/m 2 ) causes abnormalities in sexual function and sperm count with an increase in DNA fragmentation index. Possible mechanisms for fertility delay include rise in testicular temperature due to fat deposition, hyperinsulinemia, and affected hypothalamo-pituitary axis due to increased estrogen.
Smoking and alcohol: Maternal and paternal smoking and alcohol consumption habits significantly influence the success of IVF. Female alcohol consumption was found to be associated with 13% decrease in the number of eggs aspirated, if alcohol was consumed 1 year prior to IVF or gamete intrafallopian transfer (GIFT) attempt. One-month prior use increases the risk of not achieving pregnancy by 2.86 times, and 1 week before the procedure, it increases the risk of miscarriage by 2.21 times. Regarding alcohol consumption in men, it is documented that additional drink per day increases the risk of not achieving a live birth by 2.28–8.32 times; this also holds true for beer. The risk of miscarriage is increased by 2.70–38.04 times for men who drank 1 month before and during IVF and GIFT. Smoking in both females and males affects success of ARTs, but caffeine consumption has no effect on natural fertility rates. Smoking in males is found to increase the risk of pregnancy loss, while female smoking causes an adverse effect on ovarian reserve. The decrease in IVF success caused by lifestyle factors and poor nutritional habits is thought to be due to chronic oxidative stress.
Healthy lifestyles during the periconceptional period is a window of opportunity to attain successful ART outcomes. Therefore, by modifying behaviors, short-term reproductive health as well as general health can be improved.
Types of assisted reproductive technology
ARTs comprise a number of procedures including intrauterine insemination (IUI), in vitro fertilization and embryo transfer (IVF-ET), GIFT, ZIFT, and ICSI. The sequential approaches in assisted reproductive clinics are chosen from one of the following:
Intrauterine insemination (IUI): It is a type of ART in which the sperms are placed in the uterine cavity at the time of ovulation. It is a treatment of choice in a natural cycle with or without ovarian stimulation by oral antiestrogens or gonadotropins. The success of IUI and timed intercourse (TI) is almost the same.
In vitro fertilization and embryo transfer (IVF-ET): In IVF, one or more fertilized eggs (embryos) are inserted to nurture in the uterine cavity. Excess embryos are kept for future frozen embryo transfer cycles (FET). Initially, IVF was recommended only for problems in fallopian tubes. Nowadays, it is extended to include other causes like endometriosis, male factor, or unexplained infertility. IVF is the most commonly used technique and the mainstay of ART, since ET can be achieved in the same cycle, or cryopreserved embryos could be transferred in a subsequent cycle ( Table 12.1 ).
IVF (in vitro fertilization)
GIFT (gamete intrafallopian transfer)
ZIFT (zygote intrafallopian transfer)
IVF can be done in many causes of male and female infertility
Used in male factor fertility problem and unexplained fertility problem where woman has normal tubes
Used where transcervical gamete transfer is not possible
The eggs and sperms are fertilized outside the womb
The egg is fertilized in the tube
Fertilization is done in a laboratory outside the body
The zygote is transferred to the woman’s uterus
The gametes (egg and sperm) are transferred to the woman’s fallopian tubes and not the uterus
The gametes are fertilized in the laboratory, and the fertilized egg is transferred to the tube rather than the uterus
One or more embryos are drawn into a transfer catheter and placed in the uterus
Laparoscopy is mandatory to transfer the sperm and egg to the tubes
Laparoscopy is mandatory to transfer the zygote to the tubes
Fertilization confirmed when embryos are transferred
Fertilization cannot be confirmed as with IVF
Fertilization cannot be confirmed as with IVF
Most commonly used ART method
Comprises less than 1% of ART procedures
Comprises less than 1% of ART procedures
Gamete intrafallopian transfer (GIFT): It is not widely used nowadays, and technique of this is much of the same as IVF. In this procedure, eggs are collected the same way as in IVF, but are transferred laparoscopically along with the motile sperm to the fallopian tube for in vivo fertilization. It is recommended for cases, where infertility is due to an unexplained male factor.
Zygote intrafallopian transfer (ZIFT): In this procedure, embryo is developed in vitro, but is transferred laparoscopically to the fallopian tube. It is specifically used in situations, where transcervical gamete transfer is not possible. However, with the latest advancements in “in vitro” conditions supporting normal fertilization and preimplantation embryo development, GIFT and ZIFT are no longer in use.
Intracytoplasmic sperm injection (ICSI): It is a technique by which a morphologically normal and motile single spermatozoon is injected directly into the ooplasm through the process of micromanipulation. Sperm is selected on the basis of its motility and morphology for ICSI.
Assisted reproductive technology treatment cycle
An ART treatment cycle is a process through which a woman undergoes multiple steps of this procedure, including pituitary downregulation, controlled ovarian stimulation, ovulation induction, oocyte and sperm retrieval, fertilization, ET, and luteal support. The appropriate management of these steps is crucial for a good reproductive outcome of ART.
Pre-assisted reproductive technology (pre-ART) strategies
Pre-ART strategies are meant to start before ART cycle in order to improve the reproductive outcome of treatment. These strategies include the following:
Pre-ART hormonal treatment: It is for the development of follicle and endometrium recommended as per National Institute for Health and Care Excellence (NICE) guidelines. It has been reported that the chances of pregnancy increase fourfolds when a gonadotropin-releasing hormone (GnRH) agonist is used for a period of 3–6 months, especially in women with endometriosis-associated infertility.
Pre-ART surgical and other interventions: Other intervention strategies recommend the use of antioxidants, vasodilators, metformin, and surgical procedures.
Steps of ART cycle ( Fig. 12.1 )
Pituitary downregulation: This is done temporarily to suppress the pituitary gland’s release of gonadotropins and premature ovulation from early exposure to luteinizing hormone (LH). This pituitary downregulation ensures that the oocytes are available for retrieval prior to insemination in the laboratory. The downregulation protocols are selected on the basis of maternal age, ovarian reserve, BMI, past history (medical, endocrine, and fertility), preference of the clinician or center, and the patient’s previous outcome and anticipated ovarian response.