18.1 Normal Mammalian Gamete Development

Male and female gamete cells are unique because they are the only haploid cells in the body. Thus they are the only cells that go through the process of meiosis. The timing and gaps between meiotic events vary significantly for male and female gametes. A closer look at some of these differences can provide some insight as to why reproductive aging in particular has such an adverse effect on oocyte, and subsequent embryo, quality.

Male and female germ cells originate from primordial germ cells in very early embryonic development, where they develop into either oogonia in females or spermatogonia in males. In females, oogonia mitotically reproduce to a peak number around seven million cells while still in utero. A large number of these oogonia undergo atresia, while the remaining oogonia become encapsulated by follicular cells to form primordial follicles and go through the first meiotic division to become primary oocytes. At birth, a female has somewhere between 200,000 and 400,000 of these primary oocytes which are at the meiotic prophase I stage. These primary oocytes will stay at this stage until hormonal signals in puberty cause resumption of meiosis I. The primordial follicles are recruited in groups of about 50 follicles daily in a continual process through unknown mechanisms. The initial growth of these early follicles is a long process of several months, at which time most of the follicles will undergo atresia. Usually somewhere between five and ten of the early follicles will be rescued from atresia and continue to grow. The number of primordial follicles that develop, or are recruited, varies from cycle to cycle, but numbers will decrease with advancing reproductive age. Each cohort of rescued primordial follicles will resume meiosis with further nuclear and cytoplasmic development when signaled by circulating FSH levels that begin in puberty after years of senescence. Some oocytes may stay at meiotic prophase for up to 50 years! Overall, only about 400 oocytes fully mature during a woman’s reproductive life span [1].

In males, spermatogonia migrate to the fetal testes early in development, where they remain in the seminiferous tubules until puberty. While females are born with all of the oocytes that they will ever have, male spermatogonia utilize a different pathway to create gametes. A spermatogonia cell will undergo a mitotic division to produce two new cells: a primary spermatocyte and a spermatogonial stem cell. The spermatogonial stem cells allow males to continue producing sperm for most of their lives under normal circumstances. These “immortal” stem cells can explain how men who withstand testicular trauma or the use of chemotherapeutic agents may regain sperm production over time, as it only requires a small number of stem cells to survive and repopulate.

The other cell created during the mitotic division, the primary spermatocyte, will undergo meiosis I to produce two haploid secondary spermatocytes. The two secondary spermatocytes will then each undergo meiosis II and give rise to four spermatids. Immature spermatids then go through a maturation process called spermiogenesis as they progress through the male reproductive tract to morphologically differentiate into mature sperm. Thus, each spermatogonia cell eventually yields four sperm cells, allowing the testes to produce billions or trillions of sperm in a lifetime, unlike the ~400 eggs a woman will produce.

18.2 History of Intracytoplasmic Sperm Injection (ICSI)

Shortly after the initial successes of in vitro fertilization (IVF) were reported in the late 1970s [2–4], it became apparent that there was a need to broaden the indications for assisted reproductive technology (ART) treatment as it was originally applied to women with only tubal factor infertility. The role of sperm was almost taken for granted. However, as IVF became more widely adopted, it came to light that male factor issues were a major component of infertility, inviting a new focus on treating sperm issues. Male factor infertility was a major focus of study throughout the 1980s, with a great deal of attention paid to the spermatozoa and the oocyte-spermatozoan interaction.

One of the earliest indications for poor sperm quality in IVF was low concentration of sperm. Early attempts to simply concentrate the specimen, either by adding more sperm to the insemination dish or lowering the insemination volume of the culture, were ineffective [5]. Attention then turned to the oocyte, in an attempt to make it more amenable to sperm penetration. Experiments to manipulate the zona pellucida were attempted, including applying acidic Tyrode’s solution to the zona to create a hole for the sperm to enter and mechanically creating a hole or split in the zona. Unfortunately these experiments either showed very poor to no fertilization or high polyspermy rates [6–8].

The earliest successes with circumventing total fertilization failure were with a procedure involving subzonal injection of sperm into the perivitelline space of the oocyte, the area between the zona pellucida and the actual oocyte, which was referred to as SUZI [9, 10]. While attempting to refine and improve the SUZI technique, investigators in Brussels unintentionally created a new technique that would revolutionize ART treatments. The investigators noted when they were attempting to inject the sperm into the perivitelline space, they would sometimes unintentionally pierce the oolemma and inject the sperm directly into the cytoplasm of the oocyte. They noted when this “accident” occurred and were surprised to discover that these accidentally injected eggs had close to 100% fertilization rates! They began calling this procedure ICSI, which is short for intracytoplasmic sperm injection. The researchers continued to perform both SUZI and ICSI on patients and waited until they had four pregnancies from this procedure before they published the first successful series of ICSI cases in 1992 [11]. These cases used ejaculated sperm with poor semen parameters that had previously failed conventional insemination and SUZI procedures. After this fortuitous incidental discovery, further work was done to refine the ICSI technique. Subsequent studies regarding the positioning of the oocyte during injection helped create one of the most universally adopted IVF protocols used worldwide today [12–14].

The next frontier to tackle was men with no sperm in the ejaculate. ICSI has been successfully applied to patients with both obstructive and nonobstructive azoospermia [15]. For cases of obstructive azoospermia, where spermatogenesis is normal, sperm can be surgically retrieved in an outpatient procedure from either the epididymis or testes. Surgically retrieved sperm can be used fresh if coordinated with the oocyte retrieval or frozen for future use. Sperm can be extracted from the epididymis through either microsurgical sperm aspiration (MESA) or percutaneous epididymal sperm aspiration (PESA). Epididymal sperm is normally motile.

Nonobstructive azoospermia is a more difficult problem to treat due to the etiology of the azoospermia. Nonobstructive azoospermia is often due to spermatogenic failure. Failure of the testes to make significant amounts of sperm can be induced by chemotherapeutic or radiologic treatments but can also be caused by abnormal steroid hormone production or genetic defects. In such cases it is sometimes possible to find small amounts of sperm in the testes. Sperm retrieval techniques include extraction via either testicular sperm extraction (TESE) using a needle or opening the testicle(s) and using a microscope to visually dissect dilated seminiferous tubules (micro TESE) [16], which are then further dissected by laboratory staff to search for viable sperm. Sperm derived directly from the testes are typically nonmotile or weakly motile, as they have not gone through the maturation process that occurs as sperm naturally progress through the epididymis. Using nonmotile sperm is technically more challenging for embryologists as they must try to make a determination that a sperm is indeed alive although immotile. While success rates with nonmotile testicular sperm are low, the advent of ICSI made it possible to attempt pregnancy with their own genetic material in these patients.

ICSI is a physiological side step, effectively eliminating the need for motile spermatozoa or the sperm-oocyte interaction at the cell surface level. ICSI is also invasive, as it requires breakage of the oolemma and addition of an entire spermatozoan into the cytoplasm of the oocyte [14]. Due to these inherently nonphysiological interventions, it is reasonable and prudent to question the safety of ICSI. Numerous studies have examined the effects of ICSI on everything from miscarriage rates to minor and major birth defects. By design, ICSI is a procedure utilized by a cohort seeking medical intervention to achieve pregnancy. Thus, it is impossible to separate ICSI effects from infertility effects. While data on over 25 years of ICSI is mostly reassuring, there is one caveat. The potential for a male factor infertility issue, typically through a Y-chromosome microdeletion, is real. While this potential defect could cause issues for male offspring, when faced with that risk versus not having a biological child, couples overwhelmingly choose to accept this risk. Overall, the general consensus is that ICSI is an effective and safe treatment option, especially for male factor infertility [17].

18.3 Indications for ICSI

As described in the previous section, ICSI was initially developed in the early 1990s for patients who had severely abnormal semen parameters or cycles with previous total fertilization failure with conventional insemination. Since its inception, ICSI has become widely adopted for many uses including the aforementioned male factor infertility, unexplained infertility, preimplantation genetic testing, frozen gametes, serodiscordant couples, and advanced maternal age and poor responders, among others.

18.4 Diminished Ovarian Reserve (DOR) and Advanced Reproductive Age (ARA)

Age is the main limiting factor for female fertility and good reproductive outcomes. As more and more women delay childbearing until their late 30–40s [32], the need for assisted reproduction via IVF increases. Conventional stimulation protocols were historically created and honed for young women with normal to high ovarian reserve. Aging oocytes, due to a number of intrinsic and extrinsic factors, require special considerations in the IVF laboratory. Many clinics have adopted an “ICSI-all” protocol, as this can streamline both the IVF and andrology laboratory workflow when all samples for all patients are treated in the same fashion. Proponents of this approach cite evidence indicating that ICSI does not provide a disadvantage over conventional insemination when used for non-male factor infertility [33–35].

Patients with aging oocytes require special considerations in the IVF laboratory. It is well documented that disturbances in the meiotic spindle, a crucial cellular organelle comprised of spindle fibers that move and segregate chromosomes during nuclear division, can adversely affect all aspects of embryo development [36–38]. The meiotic spindle cannot be observed with the standard inverted microscope typically used for ICSI. Noninvasive polarization microscopy (i.e., PolScope) can be used in conjunction with a standard ICSI microscope to visualize meiotic spindles in oocytes. Studies with the PolScope have demonstrated that oocytes from older patients [39] and poor-responder patients [40] have lower fertilization rates and overall lower success rates than younger patients. However, PolScope use requires denudation of oocytes, hence is only useful for guidance with ICSI. Aging oocytes with substandard meiotic spindles would likely benefit from conventional insemination rather than risking further spindle disruption from ICSI.

Similarly, the competence of the oocyte regarding cytoplasmic maturation and oocyte degeneration are also factors to consider. Studies examining the ICSI technique and cytoplasmic maturation indicate that oocyte cytoplasm becomes more viscous as the oocyte reaches maturity [41, 42]. This allows penetration of the oolemma to occur without complete loss of cytoplasm, which would cause immediate oocyte degeneration. While the overall oocyte degeneration rate during ICSI is typically low, patients with fragile eggs due to DOR and/or ARA would likely benefit from conventional insemination over ICSI in the absence of male factor issues, in part to avoid potential oocyte degeneration.

Oocyte development within the ovarian follicle requires bidirectional cross talk between the cumulus cells surrounding the oocyte and oocyte itself [43]. Conventional insemination methods keep the cumulus-oocyte complex intact longer. A sibling oocyte study comparing IVF versus ICSI in patients with no known male factor issues showed a higher number of available embryos for transfer or freeze and a lower oocyte degeneration rate in conventionally inseminated oocytes [44]. A larger, retrospective study consisting of women aged 40 years and over yielded similar results, with more available embryos in the IVF group over ICSI [21].

Conventional insemination for non-male factor cases is used less and less frequently, likely due to patient pressures. If it can be done, why not? The easy answer for practitioners faced with this question is to simply move forward with ICSI. With more and more patients presenting with combinations of DOR and ARA, it may be worthwhile for physicians to build an “ICSI versus conventional insemination for DOR/ARA” module into their IVF consultation. Women with DOR/ARA may benefit from conventional insemination for several reasons. First, the cumulus-oocyte complex in maintained intact overnight, instead of denuding these cells from the oocyte within just a few hours of egg retrieval. Allowing these cells to remain with the oocyte likely allows more eggs to reach maturity and subsequently fertilize, as opposed to oocytes that are stripped for ICSI and discarded if immature. Second, ICSI is an aggressive method of fertilization with subtle but potentially significant differences between operators. For patients with advanced reproductive age, small details such as amount of aspirated oocyte cytoplasm or aggressive cumulus cell denudation may be the difference between a viable embryo or not [45]. Conventional insemination reduces the chance of oocyte degeneration due to rough handling during ICSI.

Overall, patients with diminished ovarian reserve and/or advanced reproductive age can still be suitable candidates for minimal stimulation protocols, but their care plan, especially in the IVF laboratory, needs to be carefully scrutinized. Patients who benefit from minimal stimulation protocols for ovarian stimulation will have oocytes that can benefit from minimal manipulation in the laboratory [46]. Quick and careful handling of these delicate oocytes, along with standard conventional insemination methods in the absence of a clear male factor issue can help in the long-term goal of achieving a family for this patient population.

18.5 Conclusion

Development of the ICSI procedure revolutionized the practice of IVF. ICSI is now the gold standard implemented worldwide for treatment of male factor infertility with excellent outcomes. The application of the ICSI technique has become more widespread, with uses far beyond just male factor issues. However, for DOR/ARA patients with lower quantities of potentially more delicate oocytes, it is prudent to consider conventional insemination as a first-line treatment in the absence of male factor infertility.