Round Spermatid Injection

From a fertility perspective, men with azoospermia represent a challenging patient population. When no mature spermatozoa are obtained during a testicular sperm extraction, patients are often left with limited options, such as adoption or the use of donor sperm. However, it has been reported that round spermatids can be successfully injected into human oocytes and used as an alternative to mature spermatozoa. This technique is known as round spermatid injection (ROSI). Despite the limitations of ROSI and diminished clinical success rates, the use of round spermatids for fertilization may have potential as a treatment modality for men with azoospermia.

Key points

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The likelihood of successful identification of mature spermatozoa during a microdissection testicular sperm extraction procedure performed for azoospermia is between 40% and 60%.
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Round spermatids, which are immature precursors to mature spermatozoa, are seen in approximately 30% of men with nonobstructive azoospermia without sperm seen at the time of microdissection testicular sperm extraction.
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A recent publication from 2018 reported that successful births could be achieved through the use of round spermatid injection (ROSI) and that children born from ROSI were not at an increased risk for congenital malformations.
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Concerns regarding the potential risk of abnormal epigenetic patterns following ROSI remain.
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Overall low success rates have limited the clinical application of ROSI, although improvements in the identification of round spermatids and the technique itself may lead to higher utilization in the future.

Introduction

Azoospermia affects 10% to 15% of infertile men and is defined as no sperm seen in the ejaculate in a centrifuged sample. Although patients with obstructive azoospermia are likely to have sperm retrieved with a procedure such as a testicular sperm aspiration (TESA), around 60% of men with azoospermia have nonobstructive azoospermia (NOA) and thus lower rates of successful sperm retrieval. NOA is due to defects in spermatogenesis, usually from primary testicular dysfunction. Studies have shown that the likelihood of retrieval of sperm in NOA patients during microdissection testicular sperm extraction (microTESE), the standard of care for sperm extraction in men with NOA, is between 40% and 60%. ^, Y-chromosome microdeletion is present in 3% to 15% of men with severe oligozoospermia as well as in men with NOA. In a sizable portion of azoospermic men, there is no sperm seen after microTESE, making it impossible for these men to father biologic offspring. Round spermatids are precursors of mature spermatozoa and are seen in about 30% of NOA men with no spermatozoa seen on microTESE ( Fig. 1 ). These are immature sperm cells that still contain a haploid genome, similar to the genetic composition of mature spermatozoa. Round spermatid injection (ROSI) uses this fact to inject these sperm precursors directly into an oocyte in hopes of fertilization and pregnancy.

Spermatogenesis and sperm function

Spermatogenesis is the process by which diploid spermatogonia become haploid spermatozoa ( Fig. 2 ). The spermatogonia increase in number via mitosis, and in the first stage of spermatogenesis, mitotic division results in diploid primary spermatocytes. These primary spermatocytes undergo meiosis I to form secondary spermatocytes and meiosis II to form spermatids, such as round spermatids. At this point, spermatids have the haploid genetic material that spermatozoa contain, but the spermatids are not yet motile and are not yet able to fertilize an oocyte. In the next phase, also called spermiogenesis, the round spermatids become elongated and eventually develop a tail as they progress to become mature spermatozoa. For normal fertilization to occur, the spermatozoa must provide genetic material to the oocyte by means of the centrosome and initiate oocyte activation.

History of assisted reproduction in azoospermia

Intracytoplasmic sperm injection (ICSI) was developed in the 1990s and has been revolutionary in allowing paternity for men with severe male factor infertility. In this procedure, a single spermatozoon is directly injected into the oocyte. This allows for testicular sperm extraction as an assisted reproductive technology, because sperm retrieved by these methods have not fully matured and do not yet have the ability to swim or fertilize an egg. Despite initial theoretic concerns about the long-term outcomes of children born by ICSI, any negative effects appear to be minimal, and ICSI has seen widespread use in recent years. ^, The use of testicular sperm with ICSI has allowed many men with NOA as well as men with obstructive azoospermia to achieve fatherhood and have biological offspring. Before the advent of ICSI, there were limited options for patients with severe male factor infertility. In patients without male factor infertility, the live birth rate was 36.5% with ICSI compared with 39.3% with conventional in vitro fertilization (IVF) alone. This 2015 study also found that the use of ICSI increased from 76.3% to 93.3% from 1996 to 2012 in cycles with male factor infertility present. Not only that, ICSI use increased in cycles without male factor infertility from 15.4% to 66.9% during the same time period.

Round spermatid injection in animal models

In the 1990s, there were several animal studies that reported successful births and healthy offspring via ROSI. Kimura and Yanagimachi in 1995 reported a fertilization rate of 77% and a pregnancy rate of 28.2% with healthy offspring in mice. They found that in the mouse, gamete imprinting happened before spermiogenesis. However, oocyte activation could not be triggered by spermatids, so this was done by electric current. Oocyte activation requires a soluble sperm factor, which is thought to be contained in spermatozoa’s cytoplasm; it enables oocytes to develop a characteristic series of calcium spikes that round spermatids seem to lack, but it was found that round spermatids could be treated with a calcium ionophore.

In 2011, Ogonuki and colleagues looked at fertilization of mouse oocytes using round spermatids without using artificial oocyte activation. Round spermatids in mice lack the capacity to activate an oocyte at this stage, but the investigators found when the round spermatids were frozen and thawed before microinjection, a proportion of them still developed into 2-cell embryos without artificial activation. Using frozen-thawed spermatids was thought to help with the oocyte-activating capacity in this study.

Ogonuki and colleagues in 2017 studied spermatid injection in the common marmoset using immature male marmosets. The spermatids were found to acquire the ability to activate an oocyte at the late round spermatid stage. Marmoset oocytes were then microinjected with frozen-thawed late round spermatids and were able to develop to the 8-cell stage.

Despite the feasibility of this procedure, the broad adoption of ROSI has been limited because of controversy surrounding using this beyond research purposes. In addition, it must be noted that physiologic differences in the oocyte activation process between animal models and humans may exist. Therefore, certain oocyte activation protocols and fertilization techniques, which demonstrate success in animals, may not result in successful results in humans. The issue of potentially increased rates of embryonic aneuploidy and epigenetic aberrations must also be considered in humans, whereas, in animals, these issues may have a lesser role.

Clinical use of round spermatid injection

The first report of human fertilization with spermatid injection was by Vanderzwalmen and colleagues in 1995. Tesarik and colleagues then published a case series in 1996 of 11 cases of spermatid injection, 6 with round spermatids ( Table 1 ). Fertilization occurred in 10 of 11 treatment cycles, and a pregnancy was achieved in 2 ROSI cycles, which then proceeded to live birth. However, these results were not replicated at fertility centers across the world when first attempted. Tesarik and colleagues stressed the importance of using the whole round spermatid, avoiding the use of just the nucleus. Vanderzwalmen and colleagues published a series in 1997 of 73 azoospermic men in which 260 oocytes were injected with round spermatids. Of a total of 39 transfers, 5 pregnancies were achieved with a total of 3 term births, 1 miscarriage, and 1 ongoing pregnancy. The implantation rate was 5.5%.

Table 1

Outcomes of clinical studies of round spermatid injection

Data from Refs. ^,^,^,

Author, Year	Fertilization Rate, %	Pregnancy Rate, %	Live Birth Rate, %	Oocytes Injected	Oocytes Fertilized	Embryos Transferred
Tesarik et al, 1996	35.9	16.7	16.7	39	14	12
Vanderzwalmen et al, 1997	21.9	14.3	14.3	260	57	7
Antinori et al, 1997	55.6	3.6	—	135	75	56
Antinori et al, 1997	46.7	16.7	—	15	7	6
Yamanaka et al, 1997	69.4	0.0	0.0	49	34	24
Kahraman et al, 1998	25.6	3.1	0.0	199	51	32
Barak et al, 1998	62.2	4.3	4.3	37	23	23
Bernabeu et al, 1998	44.9	0.0	0.0	69	31	31
Ghazzawi et al, 1999	22.0	0.0	0.0	574	126	40
Al-Hasani et al, 1999	18.4	0.0	0.0	49	9	9
Gianaroli et al, 1999	40.0	50.0	50.0	5	2	2
Balaban et al, 2000	56.2	—	—	356	200	—
Tesarik et al, 2000	53.8	—	—	26	14	—
Levran et al, 2000	45.5	0.0	0.0	178	81	48
Vicdan et al, 2001	28.3	0.0	0.0	69	17	5
Urman et al, 2002	40.5	0.0	0.0	1021	414	16
Sousa et al, 2002	15.9	0.0	0.0	126	20	9
Khalili et al, 2002	21.4	0.0	0.0	42	9	6
Sousa et al, 2002	34.6	—	—	26	9	—
Ulug et al, 2003	41.7	0.0	0.0	36	15	10
Tanaka et al, 2015	59.5	14.4	5.8	734	437	208
Tanaka et al, 2018	56.8	3.6	2.2	14,324	8132	3882