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Reprint requests: Gianpiero D. Palermo, M.D., Ph.D., The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, 1305 York Avenue, Y720, New York, New York 10021.
To treat couples with total fertilization failure (TFF) based on a combined oocyte- and sperm-related oocyte activation deficiency by optimizing oocyte response to chemical activation with calcium ionophore.
Design
Case report.
Setting
Tertiary Hospital.
Patient(s)
Two couples with a history of TFF after intracytoplasmic sperm injection intracytoplasmic sperm injection (ICSI).
Intervention(s)
To overcome oocyte-related oocyte activation deficiency (OAD), extended in vivo/in vitro oocyte maturation was performed to enhance ooplasmic maturity; to address sperm-related OAD, assisted gamete treatment (AGT) was performed to trigger oocyte activation.
Main outcome measure(s)
Treatment cycle outcomes for the 2 couples undergoing ICSI with extended oocyte maturation (EOM) and AGT.
Result(s)
We identified 2 couples with TFF after ICSI because of a combined factor of OAD confirmed by phospholipase C zeta expression and genomic assessment. Initial AGT treatment alone failed to enhance fertilization, suggesting superimposed oocyte dysmaturity prohibiting oocytes from responding to chemical stimuli. To address this complex form of OAD, in couple 1, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after EOM; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved initially. Two embryos were thawed and transferred, yielding a monochorionic diamniotic twin pregnancy. Couple 2 underwent 3 ICSI cycles with EOM and AGT; 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). A total of 12 blastocysts were cryopreserved. A single 46XY blastocyst was thawed and transferred, resulting in a singleton pregnancy.
Conclusion(s)
Our study has demonstrated the usefulness of EOM by targeting spindle presence to enhance chemical responses to AGT.
). This prompted efforts in assisted reproductive technology and gamete micromanipulation to improve fertilization rates and IVF success. Since the first pregnancy through intracytoplasmic sperm injection (ICSI) was reported in 1992, ICSI has become the primary mode of fertilization for virtually all forms of male factor infertility, including oligozoospermia, asthenozoospermia, teratozoospermia, and cryptozoospermia (
Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive TechnologyIntracytoplasmic sperm injection (ICSI) for non-male factor indications: a committee opinion.
ICSI enables even suboptimal spermatozoa to traverse the zona pellucida and oolemma; however, the process of oocyte activation and pronuclear formation still requires normally-functioning spermatozoa. During fertilization in vivo, phospholipase C zeta (PLCζ), a labile protein that is stored inside the perinuclear theca of the sperm cell, is deposited into the ooplasm during sperm-oocyte fusion. The PLCζ activates downstream signaling to trigger intracellular calcium ion (Ca2+) spikes that result in the resumption of meiosis and nuclear decondensation (
). Various sperm bioassays are used as adjuncts to conventional semen analyses to assess the function and competence of male gametes. These include the direct visualization of PLCζ using immunofluorescence and a confirmatory mouse oocyte activation test (MOAT)(
Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study.
Although fertilization failure is often attributed to the male gamete, oocyte-related factors should not be overlooked because a receptive ooplasm is quintessential for oocyte activation (
). The TFF may stem from ooplasmic dysmaturity because of asynchrony between cumulus-corona morphology and oocyte nuclear maturity seen in GnRH agonist stimulation (
). The process of oocyte maturation is dynamic, making it difficult to pinpoint when an oocyte is ready for fertilization. However, we believe that the assessment of the oocyte’s metaphase II (MII) spindle provides important clues. The presence and morphology of oocyte MII spindle directly reflect cytoplasmic maturity because of its high sensitivity to changes in the ooplasm, and the quantity of tubulin can be directly assessed noninvasively by light retardance of the spindle (
We present 2 cases of TFF after ICSI where initial evaluation implicated the male gamete. However, further investigation suggested a combination of sperm- and oocyte-related OAD. Subsequent treatment with extended oocyte maturation (EOM) and assisted oocyte activation improved fertilization rates and allowed both couples to achieve clinical pregnancies.
Informed consent was obtained from each couple for this case report. Studies on additional male infertility screening and gamete treatment were approved by the institutional review board of the New York Presbyterian Hospital-Weill Cornell Medicine (IRB#1006011085 and # 0712009553), and both couples gave their informed consent.
Case 1
A 31-year-old female patient with oligomenorrhea presented to our clinic with failure to conceive for 6 months. The couple initially underwent 2 cycles of ovulation induction with timed intercourse but did not conceive despite successful follicular recruitment and ovulation. A hysterosalpingogram revealed normal-appearing fallopian tubes with bilateral spill. The couple completed 3 more ovulation induction cycles, all of which were unsuccessful, before attempting IVF with ICSI.
In the first ovarian stimulation cycle, 26 oocytes were retrieved 2067 minutes (34.5 hours) after the ovulatory trigger. Oocytes were denuded 2167 minutes (36.1 hours) after the ovulatory trigger, and all 20 metaphase II (MII) oocytes were inseminated with ICSI. A fertilization assessment performed 15 hours after ICSI revealed no fertilized oocytes (2 lysed, 18 with no appearance of pronucleus). Unfertilized oocytes were reassessed, as per our routine protocol, at 4 and 6 hours after the initial assessment. This check was performed to avoid missing asynchronous pronuclei appearance as well as late fertilization.
The male partner was then evaluated for sperm-factor OAD. PLCζ assay was performed on his raw semen sample and revealed 12.9% positivity, indicating that his spermatozoa, despite normal morphology, lacked this key protein essential for oocyte activation (Fig. 1). A concurrent MOAT was performed for biological validation of sperm-factor oocyte activation deficiency (OAD). After inseminating 35 mouse oocytes with the patient's spermatozoa via piezo-actuated ICSI, only 5 (14.3%) mouse oocytes fertilized, displaying 2 pronuclei 6 hours after insemination (Fig. 2). This fertilization rate was much lower than both a fertile donor control (n=32/35, 91.4%) and a previously established normal threshold of >85% (
Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study.
). Thus, both the PLCζ assay and biologically confirmatory MOAT indicated that the male partner’s sperm had severely compromised fertilizing potential. A confirmatory whole exome sequencing revealed a frameshift mutation on the PLCZ1 gene. A TUNEL assay was also performed on the raw sample to measure sperm chromatin fragmentation and revealed a normal level of sperm chromatin fragmentation at 11.8%. This guided subsequent intervention using assisted gamete treatment (AGT) to overcome fertilization failure (
Figure 1In PLCζ staining on spermatozoa, 4′,6-diamidino-2-phenylindole (DAPI) is used to stain DNA to visualize sperm nuclei, and PLCζ is visualized by immunofluorescent staining. Positive PLCζ is indicated by the green fluorescent protein signal. PLCζ = phospholipase C zeta.
Figure 2Fertilized mouse oocyte (2PN) versus nonfertilized mouse oocyte (0PN). Noticing that the male pronucleus of human source is larger than the female pronucleus of mouse origin. The appearance of PN is accessed by time-lapse microscopy (EmbryoScope, Vitrolife, Sweden).
In their second cycle, spermatozoa were incubated in media (G-IVF Plus, Vitrolife, Sweden) supplemented with 50 mM ionomycin (I9657, Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes before ICSI. Spermatozoa with an erratic swimming pattern, indicative of the desired membrane disruption after exposure to ionomycin, were chosen for ICSI. After the immobilization of each spermatozoon, approximately 0.4 pL of ionomycin-containing media was injected into each oocyte along with a single spermatozoon. Postinjection oocytes were then incubated in Sydney IVF cleavage media supplied with 50 mM of ionomycin for 10 minutes. These oocytes were then washed thrice in G-TL media (Vitrolife, Sweden) and loaded into time-lapse microscopy slides (EmbryoSlide+, Vitrolife, Sweden). This cycle yielded 18 MII oocytes; however, only one (5.6%) oocyte fertilized normally (1 lysed, 16 with no presence of pronucleus). This embryo was cultured up to day 3 and transferred in a fresh IVF cycle but did not implant.
After a thorough evaluation of the couple’s 2 unsuccessful cycles, we considered the possibility of a concurrent oocyte-related OAD in addition to their confirmed sperm-related OAD. To achieve optimal oocyte meiotic maturity, we offered the couple another IVF cycle, this time with extended in vivo and in vitro ooplasmic maturation followed by ICSI with AGT. In the third cycle, 34 oocytes were retrieved 2169 minutes (36.2 hours) after the ovulatory trigger. Denudation was delayed to 2460 minutes (41.0 hours) after the ovulatory trigger to increase the in vitro maturation time and prolong the interaction between each oocyte and its cumulus complex (
). Spindle evaluation began 2 hours after cumulus removal and was repeated until all 27 mature oocytes exhibited MII spindle (Fig. 3) characterized by spindle light retardance of 1.6–2.4 nm and sizes of 91.6–119.4 μm2. These oocytes were deemed to have a mature cytoplasm with optimal meiotic maturity (
). The ICSI with AGT resulted in a fertilization rate of 63.0% (17/27). For this case, to reduce the attrition of prolonged embryo culture and because we suspected poor embryo development, all conceptuses were vitrified at the zygote stage. Also, cryopreservation at this developmental stage is, at times, considered in fertility preservation cases performed at our center.
Figure 3The MII oocytes with spindle complex were visualized under polarized light microscopy (Oosight, Hamilton Thorne, Beverly, Massachusetts).
Once planned with the patient, 2 zygotes were warmed and allowed to progress to day 2, reaching the 3-cell stage, and were transferred at this stage to the uterine cavity in a programmed frozen embryo transfer cycle. The early cleavage embryo replacement was decided in consideration of the extremely severe male factor infertility that often results in poor embryo development. A positive serum beta human chorionic gonadotropin (βhCG) was detected 12 days post-transfer, and a first-trimester sonogram revealed a monochorionic diamniotic twin pregnancy. At 7 weeks gestation, she transferred care to her obstetrician.
Case 2
A 37-year-old patient with a 1-year history of secondary infertility presented to our clinic for IVF and requested preimplantation genetic testing for aneuploidy (PGT-A). Their first 2 ICSI cycles resulted in TFF (0% fertilized) and low fertilization (20% fertilized), respectively. No late fertilization was observed at 4 and 6 hours after the initial assessment. Considering a potential oocyte maturity issue, the couple was advised to undergo a conventional IVF cycle to enhance in vitro maturity while exposing oocytes constantly to gametes. In their third cycle, the fertilization rate was low (10% fertilized) with conventional in vitro insemination. Disappointingly, their first 3 cycles yielded only 2 euploid blastocysts despite a total of 65 MII oocytes retrieved. The couple did not want to transfer either of their euploid 46XX blastocysts and instead opted for additional ICSI cycles after further evaluation for poor fertilization.
Initial PLCζ assessment on the raw specimen, interestingly, revealed that the majority (73.5%) of the male partner’s spermatozoa presented this protein known to be responsible for oocyte activation. Nonetheless, a concurrrent MOAT yielded a suboptimal fertilization rate of 36.4% (12/33), which was puzzling in consideration of the contradicting result. In an attempt to identify the etiology of fertilization failure, we performed a whole exome sequencing to identify key germline mutations responsible for the poor clinical outcome. We identified frameshift mutations on genes involved in spermatogenesis (PIWIL1) and centrosomal integrity (HOOK2, MAP1S), and most importantly, we discovered deletions on genes involved in calcium channel activity (CATSPER1) and fertilization (ADAM15, ADAM30, CATSPERD).
To address sperm-related OAD, we decided to proceed with AGT. In the fourth cycle, we performed AGT with calcium ionophore, as described in the prior case. However, the patient once again experienced TFF.
The patient’s fifth, sixth, and seventh cycles were executed with EOM and ICSI with AGT. Additionally, a spindle assessment was performed and confirmed the presence of an MII spindle in 10/11 (fifth cycle), 10/10 (sixth cycle), and 11/11 (seventh cycle) oocytes. Fertilization rates were superior to prior: 5/11(45.5%) oocytes fertilized in the fifth cycle, 5/10 (50%) in the sixth, and 5/10 (50%) in the seventh. These final cycles yielded a total of 12 blastocysts, 6 of which were euploid. A single euploid 46XY blastocyst was thawed and transferred in a natural frozen embryo transfer cycle, resulting in a single intrauterine pregnancy with normal sonographic parameters. The patient was discharged to an obstetrician at 7 weeks gestation.
Discussion
In these 2 cases of TFF after ICSI, both couples were initially diagnosed with sperm-factor OAD through PLCζ staining, MOAT, and DNA profiling. Based on this diagnosis, they were offered AGT in the next cycle. This treatment failed unexpectedly in both couples, leading to a hypothesized oocyte-factor OAD as well. In subsequent cycles, we used EOM to address ooplasmic dysmaturity and monitored MII oocyte spindle characteristics to time ICSI precisely. We are pleased to report that these interventions resulted in markedly improved fertilization rates, and, to date, both couples have ongoing pregnancies. These cases underscore the importance of considering both sperm and oocyte contribution to oocyte activation for patients with TFF with ICSI.
Diagnostic assessment for fertilization failure has historically been focused on the male gamete. Unlike oocytes, spermatozoa are more readily accessible for both research and clinical testing. Several candidates for cytosolic factors in human spermatozoa have been considered and finally have been identified in PLCζ (
Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study.
). Thus, immunofluorescent staining of PLCζ, corroborated by the MOAT, is often used. Moreover, in this report, as well as in a recent publication of ours, we have added a genomic assessment to identify the gene involved in the PLCζ deficiency (
). Indeed, the second case highlights the limitations of PLCζ assessment alone. Querying the entire genome allowed us to identify other genes involved in other sperm-related proteins, such as ADAM15 and ADAM30, involved in sperm-egg interaction, and CATSPER1 and CATSPERD, involved in calcium channel and motility.
To address sperm-related OAD, assisted oocyte activation is used to mimic physiologic Ca2+ changes in the oocyte cytoplasm during sperm injection. A variety of activating protocols and agents, including calcium ionophores, ionomycin, and strontium chloride, have been proposed (
). In a recent 17-year retrospective study, AOA improved fertilization, pregnancy, and live birth rates in couples with prior low and total failed fertilization, and the improvement was most pronounced in patients with sperm-related OAD based on MOAT testing (
Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study.
), and subsequently, we have seen higher fertilization and clinical pregnancy rates in cycles treated by the additional injection of ionomycin together with spermatozoa during ICSI (
Couples that experience fertilization failure refractory to AOA likely have an underlying oocyte-related issue. In the presence of normal spermatozoa function, oocyte nuclear and cytoplasmic maturity are necessary prerequisites to normal fertilization. Apart from the presence of the first polar body to indicate arrest at the MII phase (nuclear maturation), there are no validated tests to confirm the fertilization competence of an ovulated oocyte. In our reported cases, mitotic spindle assessment was performed to verify nuclear maturity and optimally time ICSI. The meiotic spindle of MII oocytes, which are pivotal to chromosomal alignment and separation, are exquisitely sensitive. Experiments with the PolScope, an orientation-independent polarized light microscope, have shown that even minor transient changes in temperature of IVF laboratory conditions can cause the spindle to disassemble, sometimes irreversibly (
). Even transient insults to spindle integrity can have a lasting influence on an oocyte’s fertilization and developmental potential. When performing spindle assessment on MII oocytes, oocytes with visualized spindles have been observed to have a higher incidence of normal fertilization using ICSI than those with nonvisualized spindles (
Cytoplasmic maturation involves complex molecular processes, including alterations in chromatin configuration, mitochondrial redistribution, cytoskeletal remodeling, and reorganization of the endoplasmic reticulum (
). In clinical practice, emphasis is placed on optimizing the yield of MII oocytes, and cytoplasmic maturity is often presumed as it tends to parallel nuclear maturity in vivo. Our previous investigations revealed that adjusting the timing of human chorionic gonadotropin (hCG) trigger, oocyte retrieval, removal of cumulus cells, and ICSI can enhance ooplasmic and nuclear maturity of each oocyte and subsequently improve fertilization in couples with a history of complete fertilization failure (
In support of the relevance of a mature optimal ooplasm, a recent study used meiotic spindle transplantation into donor ooplast to overcome failed fertilization while still allowing women to have genetically-related offspring (
). This technique offered an alternative usage of donor oocytes, not only for their mitochondria but solely for their competent cytoplasm to correct fertilization failure while allowing patients to have their own progenies.
Although the interventions presented in this study are not new individually, their combined use in both the clinical cases enlisted is novel. Indeed, we described the compounding of extending the in vivo maturation, lengthening the interval between HCG and retrieval, as well as optimizing the timing of cumulus removal as new. In addition, the AOA described was only performed after the assessment of the PLCζ on the spermatozoa and was followed by a confirmatory heterospecific MOAT. Moreover, to ensure that the oocytes had reached optimal ooplasmic and meiotic maturity, a spindle check was also performed. Finally, to identify the gene responsible for the fertilization failure, whole exome sequencing was performed. Indeed, the role of these combined interventions executed in conjunction in this report has allowed the achievement of successful fertilization in 2 couples who had previously failed fertilization with standard AOA. So, the novelty of our study is the orchestration of all the abovementioned interventions, and we believe that this manuscript will provide important information to readers regarding the treatment of difficult cases that present with unexpected and complete fertilization failure, even with ICSI. We believe that a study on spindle observation performed in a prospective manner in cases with recurrent, complete, and unexpected fertilization failure will help to pinpoint ooplasmic maturity responsible for polymerization of the meiotic spindle and therefore help to optimize the time for ICSI injection.
Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive TechnologyIntracytoplasmic sperm injection (ICSI) for non-male factor indications: a committee opinion.
Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study.
O.D. reports being on the Scientific Advisory Board for Alife Health. P.X. has nothing to disclose. A.A. has nothing to disclose. S.C. has nothing to disclose. D.G. has nothing to disclose. Z.R. has nothing to disclose. G.D.P. has nothing to disclose.
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