Abstract
Two decades have passed since the discovery of phospholipase C zeta (PLCZ1) as the sperm oocyte-activating factor. At present, there is a general consensus that PLCZ1 is responsible for triggering the calcium (Ca2+) oscillations necessary to start the oocyte activation process in mammals. One proof is that abnormal, reduced, or absent PLCZ1 in human spermatozoa leads to fertilization failure (FF) after intracytoplasmic sperm injection (ICSI). ICSI is the most effective assisted reproduction technique and enables overcoming almost all male infertility conditions. Despite fertilization rates of up to 80%, FF does occur in 1–3% of ICSI cycles, which leaves these patients with few options for obtaining genetically related offspring. Assisted oocyte activation (AOA) using Ca+2 ionophores has emerged as a useful treatment option for these patients. While AOA has been proven very beneficial for the treatment of sperm-related FF, some cases of female-related FF cannot be overcome by AOA. Therefore, the development of appropriate diagnostic tests that predict the prognosis of AOA treatment would be advantageous to improve the clinical management of these patients and shorten the time to pregnancy. The aim of this review is to provide an up-to-date overview of the genetic causes of FF after ICSI and to discuss the advantages and disadvantages of using PLCZ1 as a diagnostic marker or therapeutic molecule in comparison with currently available diagnostic tests and treatments.
Introduction
It is now generally accepted that fertilization starts when the sperm factor phospholipase C zeta (PLCZ1) is released into the oocyte (Kashir et al. 2010, Yeste et al. 2016). Twenty years after its discovery, a tremendous amount of scientific evidence indicates that the PLCZ1 protein is responsible for inducing the characteristic calcium oscillations that stimulate meiotic progression during mammalian fertilization (Saunders et al. 2002, Stein et al. 2020). When PLCZ1 enters the oocyte cytoplasm, it promotes the production of inositol 1,4,5-triphosphate (IP3), which stimulates the release of calcium (Ca2+) through its receptor (IP3R) at the endoplasmic reticulum (Yu et al. 2012, Swann & Lai 2016). These Ca2+ oscillations then activate different oocyte kinases in a time-dependent sequence that initiate the distinct pathways required for the completion of fertilization (Ducibella et al. 2002): cortical granule exocytosis by protein kinase C (PKC) (Tsaadon et al. 2008), extrusion of the second polar body and completion of the second meiotic division via Ca2+ calmodulin-dependent protein kinase II (CaMKII) (Madgwick et al. 2005), and formation of pronuclei by Mos/Mitogen-activated protein kinase inactivation (Ducibella & Fissore 2008). These processes are followed by maternal mRNA recruitment and embryo genome activation (Horner & Wolfner 2008). Deficiency of any of the sperm- or oocyte-related proteins involved in the oocyte activation process will presumably result into fertilization failure (Yeste et al. 2016).
The discovery of PLCZ1 was crucial to the understanding of the first critical phase of human reproduction, the fertilization process. Intracytoplasmic sperm injection (ICSI), the most effective assisted reproduction technology currently available, can still fail despite an overall/mean fertilization rate of 70–80% (Palermo et al. 2017). Between 1 and 3% of ICSI cycles end up with none or few oocytes fertilized, even if the couple has morphologically normal gametes (Mahutte & Arici 2003, Esfandiari et al. 2005, Bhattacharya et al. 2013). This condition is known as fertilization failure (FF) and raises great difficulties in achieving pregnancy in these patients. ICSI combined with assisted oocyte activation (AOA), the artificial induction of calcium oscillations through the use of calcium ionophores, has emerged as a beneficial treatment for patients suffering from FF (Vanden-Meerschaut et al. 2014a). While AOA has been shown to be very beneficial in patients with male factor-related FF (Bonte et al. 2019, Cheung et al. 2020), some patients with female factor-related FF will not benefit from AOA (Combelles et al. 2010, Vanden-Meerschaut et al. 2012). In this case, oocyte donation is recommended (Ferrer-Buitrago et al. 2018a). Therefore, the development of diagnostic tests to determine which gamete is responsible for ICSI failure will help clinicians to advise for the correct treatment (AOA or gamete donation) and consequently reduce the time to pregnancy. Homologous and heterologous ICSI models have been used to study sperm activation potential in patients with FF after ICSI (Heindryckx et al. 2005, Vanden-Meerschaut et al. 2013a, Ferrer-Buitrago et al. 2018a, Cardona Barberán et al. 2020). However, these tests are difficult to introduce in in vitro fertilization (IVF) clinics as they require animal facilities, specialized equipment, and operators with high expertise in piezo-driven ICSI. In this regard, the PLCZ1 factor represents a useful biomarker to distinguish between male and female-related FF (Torra-Massana et al. 2019, Cheung et al. 2020, Meng et al. 2020).
For the past decades, PLCZ1 has been the centre of attention when studying the fertilization process. It has become clear that abnormal, reduced, or absent PLCZ1 protein leads to oocyte activation failure and that one-third of men who suffer from FF after ICSI carry a mutation in the PLCZ1 gene (Escoffier et al. 2016, Dai et al. 2020). However, the increasing trend of high-throughput sequencing in infertile patients has allowed the identification of new genes causing FF after ICSI (Yatsenko & Rajkovic 2019, Sang et al. 2021). Recently, it has been reported that in males, mutations in ACTL7A and ACTL9 affect the acrosome structure, leading to PLCZ1 mislocalization and resulting in low fertilization rates after ICSI (Xin et al. 2020, Dai et al. 2021). In females, six different genes (PATL2, TUBB8, WEE2, CDC20, TLE6, NLRP5) (Alazami et al. 2015, Chen et al. 2017a, Sang et al. 2018, Wu et al. 2019, Zhao et al. 2020a, Li et al. 2021) have also been reported to cause FF after ICSI. This highlights the complexity of the fertilization process and shows the need for further research on the genetic causes of FF after ICSI.
This review aims to (i) describe the possible causes leading to FF after ICSI and provide an update on recently discovered genetic factors, (ii) outline the available diagnostic tests for FF after ICSI based on indirect and direct PLCZ1 assessment, and (iii) highlight the advantages of using recombinant PLCZ1 injection for AOA treatment, as well as the challenges it poses compared to the use of calcium ionophores.
Fertilization failure after ICSI: update on genetic causes
FF can occur even when a good number of morphologically normal gametes are used. In some of these cases, FF could be attributed to sperm head decondensation, premature sperm chromatin condensation, and sperm aster defects (Esterhuizen et al. 2002, Terada et al. 2004), as well as spindle defects and cytoplasmic immaturity in the oocyte (Swain & Pool 2008, Combelles et al. 2010). However, the most common cause of FF after ICSI is reported to be oocyte activation deficiency (OAD) associated either with molecular sperm- or oocyte-related factors (Kashir et al. 2010, Yeste et al. 2016).
Male-related fertilization failure
Male-related OAD has been largely attributed to PLCZ1 deficiency (Kashir 2020). Twenty-one different mutations in the PLCZ1 gene have been identified in male patients exhibiting FF after ICSI (Table 1) (Yoon et al. 2008, Heytens et al. 2009, Kashir et al. 2012, Escoffier et al. 2016, Torra-Massana et al. 2019, Dai et al. 2020, Mu et al. 2020, Wang et al. 2020, Yan et al. 2020, Yuan et al. 2020), explaining OAD in 30–40% of patients screened (Torra-Massana et al. 2019, Dai et al. 2020, Yan et al. 2020). Although most articles report homozygous mutations, patients with heterozygous PLCZ1 mutations can also suffer from FF (Torra-Massana et al. 2019). Most of these mutations were subjected to functional analysis by injecting the corresponding mutant complementary RNA (cRNA) into mouse MII oocytes or in vitro matured (IVM) human oocytes (Mu et al. 2020, Yan et al. 2020). These experiments revealed that the effect of each specific mutation on the oocyte activation process is different depending on the amino acid change, as well as PLCZ1 domain affected. Yet, most of them show reduced fertilization rates, as well as an abnormal calcium pattern compared to WT PLCZ1 cRNA (Heytens et al. 2009, Escoffier et al. 2016). In line with these findings, decreased and altered levels of PLCZ1 protein have also been found in patients with sperm-related OAD, with and without PLCZ1 mutations (Cheung et al. 2020).
Overview of gene mutations causing fertilization failure after ICSI.
| Male/female factor/gene | OMIM number | Function | Phenotype | Inheritance | References* |
|---|---|---|---|---|---|
| Male | |||||
| PLCZ1 | 608075 | Sperm oocyte-activating factor | FF | AR | Yoon et al. (2008), Heytens et al. (2009), Kashir et al. (2012), Escoffier et al. (2016), Torra-Massana et al. (2019), Dai et al. (2020), Mu et al. (2020), Wang et al. (2020), Yan et al. (2020), Yuan et al. (2020) |
| ACTL9 | 619251 | Correct acrosome structure formation | FF | AR | Dai et al. (2021) |
| ACTL7A | 604303 | Correct acrosome structure formation | Lower FR, EDA | AR | Xin et al. (2020) |
| SPATA16 | 609856 | Correct acrosome structure formation | Globo, FF | AR | Dam et al. (2007), Elinati et al. (2016) |
| DPY19L2 | 613958 | Correct acrosome structure formation | Globo,FF | AR | Koscinski et al. (2011), Elinati et al. (2012) |
| PICK1 | 605926 | Correct acrosome structure formation | Globo,FF | AR | Liu et al. (2010) |
| Female | |||||
| WEE2 | 614084 | Release from MII arrest | FF | AR | Sang et al. (2018), Dai et al. (2019), Yang et al. (2019), Zhang et al. (2019), Zhao et al. (2019), Zhou et al. (2019), Tian et al. (2020) |
| PATL2 | 614661 | Transcriptional repressor during oocyte maturation | OMA, FF, EDA | AR | Chen et al. (2017b), Wu et al. (2019) |
| TUBB8 | 616768 | Spindle assembly during oocyte maturation | OMA, FF, EDA | AD, AR | Feng et al. (2016), Chen et al. (2017a, 2019), Zhao et al. (2020b) |
| CDC20 | 603618 | Activator of APC/C | OMA, FF, EDA | AR | Zhao et al. (2020a) |
| TLE6 | 612399 | SCMC component | FF, EDA | AR | Alazami et al. (2015), Lin et al. (2020) |
| NLRP5 | 609658 | SCMC component | FF, EDA | AR | Li et al. (2021) |
*The references included correspond to the articles reporting mutations causing FF phenotype.
AD, autosomal dominant; APC/C, anaphase-promoting complex/cyclosome; AR, autosomal recessive; EDA, embryo developmental arrest; FF, fertilization failure; FR, fertilization rate; Globo, globozoospermia; OMA, oocyte maturation arrest; SCMC, subcortical maternal complex.
The fact that PLCZ1 mutations are not detected in all patients with FF after ICSI indicates that other male factors may be responsible for FF. Indeed, the recently published Plcz1 knock-out (KO) mouse model shows that while Plcz1 KO mouse sperm induces very low fertilization rates and is unable to provoke Ca2+ oscillations after ICSI, the Plcz1 KO mouse is able to produce offspring naturally although with lower efficiency (Hachem et al. 2017, Nozawa et al. 2018). These results suggest the existence of alternative sperm factors or independent oocyte activation mechanisms that are active when PLCZ1 is abnormal during natural fertilization.
Lately, it was reported for the first time that mutations in two actin-like sperm proteins, actin-like 7A (ACTL7A) (Xin et al. 2020) and actin-like 9 (ACTL9) (Dai et al. 2021) (Table 1), are required for the correct acrosomal structure and distribution of the PLCZ1 protein. A homozygous missense mutation in ACTL7A was detected in two brothers from a consanguineous family. After ICSI, only 50% of the oocytes were fertilized but all arrested at four to five-cell stage. When the structure of the patients’ spermatozoa was examined using transmission electron microscopy, it was observed that the acrosome detached from the nuclear envelope, resulting in a shedding and folding shape that increased the size of the perinuclear theca (PT) (Xin et al. 2020). The PT is the structure below the acrosome that surrounds sperm nuclei and where PLCZ1 localizes in fertile sperm (mostly in the postacrosomal and equatorial regions) (Grasa et al. 2008). Analysis of PLCZ1 content in affected spermatozoa revealed a lower amount of PLCZ1, and its signal disappeared from the equatorial region of PT (Xin et al. 2020). This strongly suggests that the acrosomal defects caused by ACTL7A mutations resulted in a reduced amount and abnormal localization of PLCZ1, explaining the lower fertilization rates and embryonic arrest observed in these patients. This is consistent with previously published articles showing that aberrant Ca2+ patterns during oocyte activation not only affect fertilization rates but may also have long-term effects during pre- and post-implantation development (Ozil et al. 2006). In addition, homozygous missense mutations in ACTL9 were also reported to cause male infertility-related FF in three patients which were previously diagnosed with sperm-related OAD (MOAT group 1, see section ‘Indirect PLCZ1 assessment by heterologous and homologous ICSI tests: MOAT, MOCA, and HOCA’) (Dai et al. 2021). Co-immunoprecipitation experiments showed that ACTL9 and ACTL7A signals overlapped in the acrosomal and equatorial regions of the PT in normal sperm. In contrast, mutant ACTL9 did not interact with ACTL7A, leading to abnormal PT structure and acrosomal detachment, as observed in patients with ACLT7A mutations (Dai et al. 2021). In addition, PLCZ1 was also absent or abnormally localized in the sperm from the patients with ACTL9 mutations. Finally, both Actl7a and Actl9 knock-in mice showed the same altered acrosome structure as observed in the patients, but a more severe FF phenotype, as none of the oocytes injected by ICSI could be fertilized (Xin et al. 2020, Dai et al. 2021).
A last example of genes causing male infertility-related FF are spermatogenesis‐associated 16 (SPATA16) (Dam et al. 2007, Elinati et al. 2016), DPY‐19‐like 2 (DPY19L2) (Koscinski et al. 2011, Elinati et al. 2012), and protein interacting with C kinase 1 (PICK1) (Liu et al. 2010) (Table 1). Homozygous mutations in these three genes have been detected in patients with globozoospermia, an extreme form of teratozoospermia in which the spermatozoa have a globular shape with an altered or absent acrosome. Globozoospermic males generally have low fertilization rates after ICSI, which are also associated with abnormal and reduced PLCZ1 content caused by the altered acrosome structure (Heytens et al. 2009, Kashir et al. 2010, Taylor et al. 2010).
Apart from PLCZ1, all male genes discovered so far seem not to be involved in alternative oocyte activation pathways but rather in the correct formation of the acrosomal structure. When mutated, these factors indirectly affect PLCZ1 content and localization, ultimately leading to FF after ICSI (Elinati et al. 2012, Xin et al. 2020, Dai et al. 2021). This confirms the important and central role that PLCZ1 plays during oocyte activation and fertilization.
Female-related fertilization failure
The possible attribution of female factors to OAD has been less evident for a while. Female factors are more complex to study because oocytes are scarce for research purposes. However, whole-exome sequencing (WES) in patients with various infertile phenotypes, mainly oocyte maturation arrest (OMA), FF, and embryo developmental arrest (EDA), has revealed multiple female genes participating in the fertilization process (Yatsenko & Rajkovic 2019, Sang et al. 2021).
The clearest evidence of an oocyte factor causing FF is the protein wee1-Like protein kinase 2 (WEE2) (Sang et al. 2018). WEE2 is an oocyte kinase involved in the release from methaphase II (MII) arrest and acts downstream of Ca2+ oscillations. WEE2 inhibits cyclin-dependent kinase 1, contributing to metaphase promoting factor degradation to enable meiotic progression (Oh et al. 2011). Homozygous mutations in WEE2 have been detected in patients with low and complete fertilization failure after ICSI (Sang et al. 2018, Zhang et al. 2019, Zhao et al. 2019, Zhou et al. 2019, Tian et al. 2020). AOA failed to solve FF in these patients (Dai et al. 2019, Yang et al. 2019), but when WT WEE2 cRNA was injected into the oocytes of a patient with a WEE2 mutation, normal fertilization rate and blastocysts were obtained (Sang et al. 2018), which is in line with the predicted function of WEE2, downstream of Ca2+ signalling.
Research in patients suffering from OMA has identified mutations in three different oocyte genes: PAT1 homolog 2 (PATL2), which is a transcriptional repressor mainly involved in germinal vesicle (GV) arrest; tubulin beta class 8 (TUBB8), a β-tubulin involved in spindle assembly mostly causing metaphase I (MI) arrest; and cell division cycle 20 (CDC20), which is an activator of the anaphase-promoting complex/cyclosome (APC/C) during both mitosis and meiosis and causes GV and MI arrest. Genetic screening of these factors in a large sample population revealed phenotypic variability. Mutations in PATL2 (Chen et al. 2017b, Wu et al. 2019), TUBB8 (Feng et al. 2016, Chen et al. 2017a, Zhao et al. 2020b) and CDC20 (Zhao et al. 2020a) were also found in patients with lower fertilization rates after ICSI and impaired embryo development (Table 1). In addition, mutations in some components of the subcortical maternal complex (SCMC) have also been detected in patients suffering from FF after ICSI: transducin-like enhancer of split 6 (TLE6) (Alazami et al. 2015, Lin et al. 2020) and NLR family, pyrin domain-containing 5 (NLRP5) (Li et al. 2021) (Table 1). However, most mutations detected in the factors that form the SCMC result in impaired embryonic development (Wang et al. 2018), as the SCMC is involved in embryo genome activation and is thus critical for embryo progression during the first divisions up to the eight-cell stage. As described, oocyte factors linked to FF after ICSI are not only specific to the oocyte activation process (except for WEE2) but also participate before and after in a complex and interconnected process involving oocyte maturation, fertilization, and preimplantation embryo development. Further WES studies are needed in patients with extreme IVF outcomes to discover new genes that cause specific infertility phenotypes (Capalbo et al. 2021), such as FF. The discovery of monogenic traits causing OMA, FF, and EDA will help in the development of more appropriate treatment options for these couples.
PLCZ1 assessment as diagnostic tool to predict efficacy of AOA treatment
When a couple presents with FF after ICSI, few treatment options other than AOA or oocyte donation remain to achieve pregnancy. To predict the usefulness or the prognosis of AOA treatment in the next ICSI cycle, several diagnostic tests have been developed to identify the responsible gamete causing FF. These tests indirectly or directly assess the presence of PLCZ1 and thus the ability of the patient’s sperm to initiate the oocyte activation process. If it is confirmed that the male gamete is functional, the female gamete is considered the cause of the FF (Cardona Barberán et al. 2020).
Indirect PLCZ1 assessment by heterologous and homologous ICSI tests: MOAT, MOCA, and HOCA
Heterologous and homologous ICSI assays can be used to assess the activation rate, as well as the Ca2+ oscillatory capacity of human sperm after injection into mammalian oocytes of other species (e.g. mouse, rabbit, and bovine) and human oocytes, respectively. The mouse model is the preferred option for heterologous ICSI tests because a high yield of oocytes is easily obtained and the housing and handling of this species is easier compared to other mammals. The mouse oocyte activating test (MOAT) consists of injecting patient sperm into mouse oocytes and assessing the activation rate (two-cell formation rate) the next day after performing ICSI (Rybouchkin et al. 1995, Araki et al. 2004, Heindryckx et al. 2005). Depending on the percentage of activation rate, patients are classified into the following groups: MOAT group 1 (0–20% activation rate), MOAT group 2 (20–84% activation rate), or MOAT group 3 (85–100% activation rate, which correlates with the activation range of fertile control spermatozoa). MOAT 1 patients are diagnosed as sperm-related OAD, MOAT 2 patients as decreased sperm activation capacity, while MOAT 3 patients as suspected-oocyte-related OAD. The mouse oocyte calcium analysis (MOCA) is a more sensitive diagnostic test than the MOAT, as it allows better discrimination between sperm- and oocyte-related deficiencies in MOAT group 2. The MOCA examines the ability of human sperm to induce Ca2+ oscillations after injection into mouse oocytes (previously exposed to a Ca2+-sensitive fluorescent dye) (Vanden-Meerschaut et al. 2013a). After ICSI, the oocytes are excited in an inverted epifluorescent microscope at different wavelengths. The emitted fluorescence recorded over time (over a period of 2 h) corresponds to the Ca2+ pattern expressed by each oocyte. To determine if this pattern is normal, the average product of the mean frequency (F) and mean amplitude (A) of all oocytes injected with the patient sperm is calculated and compared to the A × F obtained in oocytes injected with control sperm. An A × F ≤ 9 indicates decreased Ca2+ induction capacity of patient sperm and thus sperm-related activation deficiency, whereas an A × F ≥ 9 product indicates normal Ca2+ release and thus, suspected-oocyte-related activating deficiency (Vanden-Meerschaut et al. 2013a). The MOCA test confirms that patient sperm from MOAT group 2 suffers from reduced activating capacity, while MOAT group 3 shows mostly normal calcium pattern similar to the pattern observed in sperm from fertile controls (Table 2). Mouse heterologous ICSI tests are valuable in predicting the response to ICSI-AOA treatment (Heindryckx et al. 2008, Bonte et al. 2019). It has been reported that AOA increases fertilization rates up to 70, 63, and 57% in MOAT group 1, group 2, and group 3, respectively. For MOAT group 3, the increase in fertilization rates is significantly lower than for MOAT group 1 and 2, implying that AOA treatment is not helpful for a part of oocyte-related factors and that oocyte donation might be recommended when ICSI-AOA fails (Bonte et al. 2019) (Table 2). Although mouse heterologous ICSI tests are advantageous to distinguish between sperm and oocyte factors, these tests cannot be performed in all IVF laboratories due to the need of mouse facilities, specialized equipment, as well as ethical concerns. In this context, the use of vitrified-thawed mouse oocytes has been proposed as a solution (Bonte et al. 2020).
Correlation of MOAT, MOCA, and HOCA diagnostic test results with ICSI-AOA outcomes. Reproduced from Cardona Barberán et al. (2020) with permission.
| MOAT group (OA rate, %) | MOCA (A × F value) | HOCA (A × F value) | Diagnosed OAD (sperm or oocyte factor) | ICSI-AOA outcome (FR and LBR) |
|---|---|---|---|---|
| MOAT 1: 0–20% | <9: absence or very abnormal number of Ca2+ oscillations | <0.6: absence of Ca2+ oscillations | Sperm-related OAD | AOA very beneficial (restores FR to normal values ~70% and increases LBR) |
| MOAT 2: 21–84% | <9: abnormal number of Ca+2 oscillations | <0.6: absence or very abnormal number of Ca2+ oscillations | Sperm-related OAD | AOA very beneficial (restores FR to normal values ~70% and increases LBR) |
| >9: normal number of Ca2+ oscillations | <0.6: absence or very abnormal number of Ca2+ oscillations | Diminished sperm activating capacity not detected by mouse assays | AOA beneficial (significantly increases FR to ~60% and improves LBR) | |
| MOAT 3: 85–100% | >9: normal number of Ca2+ oscillations | <0.6: absence or very abnormal number of Ca2+ oscillations | Diminished sperm activating capacity not detected by mouse assays | AOA beneficial (significantly increases FR to ~50% and improves LBR) |
| >0.6: normal number of Ca2+ oscillations | Normal sperm activating capacity, thus oocyte-related OAD | ICSI-AOA outcome very variable. When ICSI-AOA fails to restore FR, patients must be advised for oocyte donation. |
A, amplitude; F, frequency; FR, fertilization rate; LBR, lives birth rate; OA, oocyte activation; OAD, oocyte activation deficiency.
A more reliable and sensitive test is the recently developed human oocyte calcium analysis (HOCA) (Ferrer-Buitrago et al. 2018a). The HOCA will assess the Ca2+ oscillatory capacity of human sperm after injection into human IVM oocytes (GV or MI) or human in vivo matured MII oocytes with smooth endoplasmic reticulum aggregates (SERa oocytes) (Ferrer-Buitrago et al. 2018a). This assay is performed analogously to MOCA, but Ca2+ oscillations are examined over a 10-h period, as Ca2+ release during human fertilization takes longer. Following Ca2+ analysis, the oocytes are destroyed before the fertilization process is completed (16–18 h after ICSI). In this case, a product of A × F ≤ 0.6 indicates a sperm-related problem and a favourable response to ICSI-AOA, while a product A × F ≥ 0.6 indicates a sole oocyte-related activation deficiency and unfavourable response to ICSI-AOA (Ferrer-Buitrago et al. 2018a). Human PLCZ1 has been shown to be more potent than mouse PLCZ1 (Nomikos et al. 2014). This means that a lower amount of human PLCZ1 is sufficient to activate mouse oocytes, compared to human oocytes. The HOCA assay can therefore confirm the inability of human sperm to induce calcium oscillations in IVM oocytes for MOAT 1 and MOAT 2. However, for some patients diagnosed as MOAT 3, HOCA can reveal a diminished sperm activating capacity, making it a more sensitive test (Nikiforaki et al. 2014, Ferrer-Buitrago et al. 2018a) (Table 2). Thus, although the homologous test HOCA is a better diagnostic tool than MOAT and MOCA, IVM/SERa human oocytes are scarce for research purposes, and this test cannot be performed for all patients with FF after ICSI. Therefore, a combination of MOAT, MOCA, HOCA, and genetic screening (see ‘Direct PLCZ1 assessment: PLCZ1 genetic screening and protein quantification assays’ section) is advisable to identify the responsible gamete causing FF after ICSI, as well as to predict the probability of becoming pregnant after ICSI-AOA (Table 2). A flow diagram on the recommended order of diagnostic tests and treatment advice in couples with recurrent FF after ICSI is shown in Fig. 1. For illustrations on the detailed MOAT, MOCA, and HOCA procedures refer to Cardona Barberán et al. (2020).
Flow diagram on the order of recommended diagnostic tests and treatment advice in couples with FF after ICSI. When couples present a story of recurrent total fertilization failure or low fertilization (fertilization rate ≦ 33.3%) after ICSI, the MOAT test is offered at the IVF laboratory from Ghent University Hospital. Patients diagnosed as MOAT 1 are confirmed to have an abnormal sperm activating deficiency and thus favourable outcome after ICSI-AOA treatment. For MOAT 2 patients, the MOCA test helps to confirm the presence of sperm factor (A × F ≦ 9) and consequently ICSI-AOA is recommended. In few cases, MOCA can result in an inconclusive result (A × F > 9) meaning suspected-oocyte factor or diminished sperm activating capacity not detected by mouse assays. For these cases, as well as for MOAT 3 patients (diagnosed as suspected-oocyte factor), the HOCA test shows higher sensitivity in predicting the responsible gamete of the FF. Abnormal HOCA result confirms the presence of sperm factor and is associated with favourable outcome after ICSI-AOA. *However, FF can still occur after ICSI-AOA (in cases with sperm factor) pointing to a combined factor. For these patients, treatment advice follows oocyte donation with ICSI-AOA. Normal HOCA result (normal sperm activating capacity) indicates suspected oocyte factor and unfavorable outcome after ICSI-AOA. In these cases, the decision to undergo ICSI-AOA treatment falls on the clinician and the specific IVF story of the patient. If FF after ICSI-AOA occurs, treatment advise follows oocyte donation with conventional ICSI (Ferrer-Buitrago et al. 2018a). Alternatively, PLCZ1 genetic screening can be offered in first place. Patient with PLCZ1 mutations will show favourable outcome after ICSI-AOA. However, if the patient does not present a mutation in PLCZ1, the causal gamete is not identified and the MOAT test should be offered. aLow fertilization after ICSI is considered when fertilization rate is 33.3% or less, based on the fifth percentile of mean fertilization rates (Vanden-Meerschaut et al. 2012). TFF, total fertilization failure; LF, low fertilization; FR, fertilization rate; PLCZ1, phospholipase C zeta 1; ICSI, intracytoplasmic sperm injection; AOA, assisted oocyte activation; MOAT, mouse oocyte-activating test; MOCA; mouse oocyte calcium analysis; HOCA, human oocyte calcium analysis; A × F, amplitude × frequency.
Citation: Reproduction 164, 1; 10.1530/REP-21-0387
Direct PLCZ1 assessment: PLCZ1 genetic screening and protein quantification assays
The discovery of the PLCZ1 protein as the main sperm oocyte-activating factor (SOAF) has allowed the development of diagnostic tests based on the evaluation of abnormal PLCZ1 presence. In particular, genetic screening of PLCZ1 has emerged as an accessible and useful analysis that identifies abnormalities in the PLCZ1 sequence in 30–40% of the FF patients screened (Torra-Massana et al. 2019, Yan et al. 2020). When these patients are treated with ICSI-AOA, the fertilization rate is restored to normal values and live birth can be achieved (Torra-Massana et al. 2019, Mu et al. 2020). Quantitative real-time PCR has also been used to detect the expression of PLCZ1 mRNA in sperm samples. Although this assay identifies lower PLCZ1 mRNA expression in patients with earlier FF after ICSI compared to individuals with high fertilization rates (>70%), the exact correlation between PLCZ1 mRNA and protein levels is not revealed (Aghajanpour et al. 2011). Therefore, methods for quantifying PLCZ1 protein, such as immunostaining and immunoblotting, are a better approach and have been widely used to study the localization pattern and expression level of PLCZ1 in sperm cells. The absence of PLCZ1 protein in the equatorial region of sperm heads and reduced PLCZ1 expression are observed in patients with FF after ICSI (Heytens et al. 2009, Escoffier et al. 2016, Dai et al. 2020). In addition, the proportion of sperm with PLCZ1 in a patient sample has been shown to correlate with fertilization rates following ICSI (Yelumalai et al. 2015, Kashir et al.2020) and has therefore been proposed as a biomarker for predicting the response to ICSI-AOA (Nazarian et al. 2019, Cheung et al. 2020, Meng et al. 2020). The PLCZ1 test proposed by Meng et al. (2020) consists of classifying FF patients according to the mean PLCZ1 expression in spermatozoa and the proportion of sperm exhibiting PLCZ1 compared to the control group. Patients are diagnosed as: PLCZ1 deficient (when both parameters are significantly lower than in the control group), PLCZ1 reduced (when one of the parameters is significantly lower than in the control group), or PLCZ1-normal (when no significant difference in found in either parameter). The cut-off values for PLCZ1 deficiency were determined to be 15.57 a.u. for the PLCZ1 level and 71% for the percentage of sperm containing PLCZ1. Of the 43 patients studied, 15 were classified as PLCZ1 deficient and 17 as PLCZ1 reduced. In five PLCZ1-deficient patients who underwent ICSI-AOA, fertilization rates were increased to normal levels and four of them achieved live births (Meng et al. 2020). Cheung et al. (2020) also suggested the use of PLCZ1 immunostaining to identify the responsible gamete causing FF after ICSI. If PLCZ1 is present in <30% of the patient sperm cells (at least 200 cells are analyzed), the FF is attributed to sperm-related OAD, whereas if PLCZ1 is present in ≥30% of the patient sperm cells, the couple is diagnosed with oocyte-related OAD. In this study, the MOAT test confirmed the sperm activating deficiency in the patients diagnosed with PLCZ1 absence. Consequently, if the patient suffered from sperm-related OAD, AOA was offered in the next ICSI cycle. An increase in the fertilization rate from 9.1 to 42.1% was observed, and 6 out of 24 patients treated with AOA achieved a live birth (Cheung et al. 2020). Although promising, the proposed PLCZ1 assays should be used with caution and with reproducible control results, as the use of polyclonal anti-PLCZ1 antibodies has led to some controversy between authors (Kashir et al. 2013). Recently, it has been shown that the use of antigen unmasking/retrieval protocols leads to better visualization of PLCZ1 fluorescence (Kashir et al. 2017).
Not surprisingly, patients with PLCZ1 deficiency respond very well to ICSI-AOA treatment, because altered PLCZ1 protein cannot correctly induce the Ca2+ oscillations required for completion of the fertilization process (Yoon et al. 2008). Therefore, diagnostic tests based on direct PLCZ1 assessment are valuable for predicting the success of ICSI-AOA treatment and might be more easily introduced in IVF laboratories than homologous/heterologous ICSI tests. However, assessment of PLCZ1 alone may underestimate the presence of other important male genes for fertilization. Besides, mutations in several female factors (WEE2, TLE6, PATL2, TUBB8, CDC20, and NLRP5) are now known to also cause FF after ICSI (Yatsenko & Rajkovic 2019, Sang et al. 2021). Therefore, creating targeted gene panels for couples with FF would be more beneficial than focusing exclusively on the PLCZ1 male factor.
AOA treatment: chemical agents or human recombinant PLCZ1 injection?
The purpose of AOA treatment after ICSI is to induce sufficient Ca2+ release in the patient’s oocytes to achieve a normal fertilization rate and downstream embryonic development. This induction of artificial Ca2+ oscillations can be achieved by different methods: by applying an electrical field to the oocytes (Mansour et al. 2009), by a vigorous aspiration of the cytoplasm during ICSI (Ebner et al. 2004), or by using different chemical agents (Yanagida et al. 2006, Heindryckx et al. 2008, Ebner et al. 2015a). Chemical agents result in higher fertilization rates and are therefore the preferred choice for AOA treatment. Among the available agents, Ca+2 ionophores (ionomycin and calcimycin) are the most commonly used (Vanden-Meerschaut et al. 2014a). Some reports describe the use of strontium chloride (SrCl2) as well as a potent chemical activation method. While Ca+2 ionophores induce single Ca+2 transients, SrCl2 generates multiple Ca+2 oscillations, at least in mouse, similar to the calcium changes during normal fertilization (although the latter has not been proven in human oocytes) (Ferrer-Buitrago et al. 2018b).
Exposure of oocytes to Ca2+ ionophores allows Ca2+ influx by permeabilization of the plasma membrane or by acting directly on IP3Rs present on the membrane of the endoplasmic reticulum (Nikiforaki et al. 2016). Both ionomycin and calcimycin (GM508 or A23187) have shown to restore fertilization and embryonic development in patients with FF after ICSI, leading to numerous live births (Ebner et al. 2015a, Bonte et al. 2019). However, when comparing the efficiency of both ionophores, ionomycin resulted in higher Ca2+ release and consequently higher fertilization rates, especially in human oocytes (Nikiforaki et al. 2016). In contrast, exposure of mouse oocytes to SrCl2 induces Sr2+ influx through the transient receptor potential cation channel, subfamily V, vanilloid 3 (TRPV3), which promotes downstream oscillations in [Ca2+]i/[Sr2+] mainly by sensitizing IP3Rs (Carvacho et al. 2013). Although SrCl2 is the most efficient method for inducing oocyte activation in mouse oocytes, its efficacy in human oocytes is still debated (Lu et al. 2018). Some smaller case series have reported live births after the use of SrCl2 in patients with FF after ICSI (Yanagida et al. 2006, Kyono et al. 2008). However, regardless of the presence of functional TRPV3 receptors in human oocytes, other authors have not detected Ca2+ oscillations or fertilization after SrCl2 exposure (Lu et al. 2018, Storey et al. 2021). Recently, this contradiction was shown to be due to the differential sensitivity of IP3R to mediate Ca2+ release between mouse and human oocytes. It has been described that the ability of Sr2+ to trigger IP3R-induced Ca2+ release depends on the content of cytosolic ATP present in oocytes. Mouse oocytes contain more ATP molecules that can modulate IP3R-induced Ca2+ release, whereas cytosolic ATP levels in human oocytes are insufficient to stimulate IP3R-induced Ca2+ release after SrCl2 exposure (Storey et al. 2021). Methods to promote ATP production in human oocytes could allow multiple Ca+2 oscillations in response to Sr2+ and thus elicit a more physiological Ca+2 response than Ca+2 ionophores. However, there are concerns about the use of non-native chemical agents as these molecules could have deleterious effects on gene expression and embryonic development (Van Blerkom et al. 2015). Therefore, injection of recombinant human PLCZ1 protein (rhPLCZ1) was proposed as an endogenous and safe AOA approach. This was based on articles showing that injection of human PLCZ1 cRNA can induce oocyte activation in both mouse (Cox et al. 2002) and human oocytes (Rogers et al. 2004). Since injection of PLCZ1 cRNA might be hazardous as it could induce uncontrolled expression of the PLCZ1 protein, several laboratories work on the production of a stable and active rhPLCZ1 protein (Vanden-Meerschaut et al. 2014a).
To date, three authors have reported the production of rhPLCZ1 in mammalian (Kashir et al. 2011) or bacterial (Yoon et al. 2012, Nomikos et al. 2013) cells and its delivery to human oocytes. The first approach did not use pure rhPLCZ1 protein but whole cell lysates of mammalian cells transfected with PLCZ1 WT protein. Injection of these cell lysates into mouse oocytes resulted in multiple Ca2+ oscillations (Kashir et al. 2011). The other two approaches produced active purified rhPLCZ1 in Escherichia coli and both demonstrated the ability of the protein to induce Ca2+ oscillations in human IVM oocytes, albeit with different efficiencies (Yoon et al. 2012, Nomikos et al. 2013). Nomikos et al. (2013) reported a greater ability to produce more physiological Ca2+ oscillation patterns at lower rhPLCZ1 concentrations. Moreover, the same study reported that failed mouse egg activation after injection of mutant human PLCZ1 cRNA could be rescued by subsequent injection of rhPLCZ1, leading to successful embryonic development up to the blastocyst stage (Nomikos et al. 2013). Similarly, Sanusi et al. (2015) described that rhPLCZ1 was able to overcome oocyte activation failure in a mouse model of failed fertilization (heat-treated sperm) and achieved significantly higher blastocyst rates than with ionomycin treatment (Sanusi et al. 2015). Lastly, a recent study using Plcz1-null mouse sperm compared different AOA strategies (i.a. SrCl2, ionomycin, and rhPLCZ1) and found that these methods yielded comparable fertilization rates, but SrCl2 produced the highest blastocyst formation. Ionomycin and rhPLCZ1 resulted in similar blastocyst formation (50% vs 41%), supporting the use of rhPLCZ1 as an appropriate AOA strategy for human oocytes (Ferrer-Buitrago et al. 2020).
Although injection of rhPLCZ1 protein seems promising as an AOA strategy, no further articles have been published on the production of rhPLCZ1 since 2013, and the protein is not commercially available. This could be explained by the difficulty to purify and obtain a stable rhPLCZ1 protein. Moreover, further research on the best clinical protocol to administer rhPLCZ1 should be conducted. Thus, Ca+2 ionophores currently remain the most commonly applied AOA method in the clinics, even though they do not mimic the physiological Ca2+ response but only induce one or two Ca2+ peaks (Nikiforaki et al. 2016). Studies conducted in mouse have shown no differences in gene expression profiles between blastocysts derived from in vivo fertilization, ICSI, and AOA using rhPLCZ1, SrCl2, and ionomycin (Ferrer-Buitrago et al. 2020), as well as no abnormalities have been detected in pups born after AOA using SrCl2 and ionomycin (Vanden-Meerschaut et al. 2013b). Moreover, these pups showed normal fertility as they mated and obtained healthy offspring (Vanden-Meerschaut et al. 2013b). Nonetheless, caution is required as few articles have been published on the health of children born after the use of human ICSI-AOA (Takisawa et al. 2011, Vanden-Meerschaut et al. 2014b, Mateizel et al. 2018, Long et al. 2020). However, these articles report on the health of the children up to age of 10 (Vanden-Meerschaut et al. 2014b). To date, no major abnormalities have been detected, but long-term follow-up studies are currently lacking, so possible long-lasting effects cannot be ruled out, although animal safety studies are reassuring. In addition, the application of AOA to couples with genetic mutations, as PLCZ1 mutations, do not prevent the transmission of the genetic alteration. Male children born with PLCZ1 mutations will also be in need to use AOA to reproduce. Thus, to avoid the use of AOA, preimplantation genetic testing for monogenic disorders could be applied to select embryos free of mutations, as it is often done when performing conventional ICSI in patients carrier of single gene defects. Finally, AOA has recently been proposed for other infertility conditions, such as embryonic developmental arrest (Ebner et al. 2015b), polycystic ovarian syndrome, advanced maternal age, or primary ovarian insufficiency (Lv et al. 2020). Although AOA may be beneficial in infertility conditions caused in part by abnormal calcium release, its use in the clinic should be reserved for patients with FF after ICSI, especially those with PLCZ1 deficiency (Cardona Barberán et al. 2020).
Conclusion
FF after ICSI remains a challenge for IVF clinics. Heterologous (MOAT and MOCA) and homologous ICSI tests (HOCA) have been used by some laboratories to distinguish between sperm and oocyte-related FF and accordingly to predict the success of subsequent AOA treatment (Heindryckx et al. 2008, Vanden-Meerschaut et al. 2013a, Ferrer-Buitrago et al. 2018a, Bonte et al. 2019). Although they have a high predictive potential for identifying sperm-related deficiencies, and thus justify the use of AOA, these assays are technically difficult and require animal facilities and specialized equipment. In this context, the discovery of PLCZ1 as the major SOAF has paved the way for the design of new and simpler diagnostic tools. PLCZ1 is now used as biomarker mainly for genetic screening and protein quantification (Torra-Massana et al. 2019, Cheung et al. 2020, Meng et al. 2020). When a patient is diagnosed with PLCZ1-deficiency, it is known that the low fertilization rates after ICSI are caused by inadequate Ca+2 release and AOA will be highly efficient. However, assays based on direct PLCZ1 assessment may omit the presence of other important factors involved in the oocyte activation process. High-throughput sequencing techniques in infertile patients have revealed new male (ACTL7A and ACTL9) and female genes (WEE2, TLE6, PATL2, TUBB8, CDC20,and NLRP5) also causing FF after ICSI (Xin et al. 2020, Dai et al. 2021, Sang et al. 2021). Until now, the male genes discovered are not involved in an alternative oocyte activation pathway but in the formation of the acrosome, which indirectly affects the localization and concentration of PLCZ1 and thus its ability to trigger calcium release (Xin et al. 2020, Dai et al. 2021). Nevertheless, targeted genetic screening panels or WES will become the preferred diagnostic tool as they can reveal more oocyte-activating deficiencies and allow the development of more precise and personalized treatments (Capalbo et al. 2021).
Finally, PLCZ1 has also been proposed as a therapeutic agent. Three laboratories have developed various human rPLCZ1 proteins and have shown that injection of this molecule results in multiple Ca+2 oscillations resembling the physiological calcium response (Kashir et al. 2011, Yoon et al. 2012, Nomikos et al. 2013). However, it is not yet commercially available and its development has been halted for several years, probably because of the difficulty in purifying a stable molecule. In the meantime, AOA with calcium ionophores remains the best strategy. Yet, AOA should only be applied for patients with clear FF, unless more data support its efficacy for other infertility conditions (Cardona Barberán et al. 2020).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was funded by the Research Foundation Flanders (FWO): no. FWO.OPR.2015.0032.01 and no. 1298722N.
Author contribution statement
A C B, A B, B H were responsible of conceptualization. A C B was responsible of literature search, making the tables and writing the original draft preparation. A B, F V M and B H were responsible of supervising the manuscript. B H was responsible of final editing and approval. All authors have read and agreed to the published version of the manuscript.
Acknowledgement
The authors thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant.
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