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Identification and treatment of men with phospholipase Cζ–defective spermatozoa

Open AccessPublished:July 23, 2020DOI:https://doi.org/10.1016/j.fertnstert.2020.04.044

      Objective

      To identify and treat the gamete responsible for complete fertilization failure with intracytoplasmic sperm injection (ICSI) using a newly proposed assisted gamete treatment (AGT).

      Design

      Prospective cohort study.

      Setting

      Center for reproductive medicine.

      Patient(s)

      One-hundred and fourteen couples with an adequate number of spermatozoa for ICSI and a fertilization rate of ≤10%, after controlling for maternal age.

      Intervention(s)

      Couples with an oocyte-related oocyte activation deficiency (OAD) underwent a subsequent cycle with a modified superovulation protocol; couples with sperm-related OAD had an additional genetic and epigenetic assessment to identify mutations and expression levels of the corresponding genes.

      Main Outcome Measure(s)

      Treatment cycle outcome for couples undergoing ICSI with either a modified superovulation protocol or AGT compared with their historical cycle.

      Result(s)

      A total of 114 couples matched the inclusion criteria, representing approximately 1.3% of the total ICSI cycles performed at our center, with age-matched controls. Fifty-two couples were confirmed negative for sperm-related OAD by the phospholipase Cζ (PLCζ) assay, indicating oocyte-related factors in their failed fertilization cycles. Couples were treated by one of two AGT protocols, AGT-initial or AGT-revised, in a subsequent attempt that was compared with their historical cycle. Subsequent ICSI cycles with a tailored superovulation protocol yielded significantly higher fertilization (59.0% vs. 2.1%) and clinical pregnancy (28.6% vs. 0) rates. In 24 couples (mean ± standard deviation: maternal age, 35.6 ± 5 years; paternal age, 39.8 ± 6 years) sperm-related OAD was confirmed; in four men, a deletion on the PLCZ1 gene was identified. Additional mutations were also identified of genes supporting spermiogenesis and embryo development (PIWIL1, BSX, NLRP5) and gene deletions confirming a complete absence of the subacrosomal perinuclear theca (PICK1, SPATA16, DPY19L). Subsequent AGT treatment provided higher fertilization (42.1%) and clinical pregnancy (36% vs. 0%) rates for couples with a history of impaired (9.1%) fertilization. A comparison of the two AGT protocols, AGT-initial or AGT-revised, revealed that the latter yielded even more favorable fertilization (37.6% vs. 45.9%) and clinical pregnancy (21.1% vs. 83.3%) rates.

      Conclusion(s)

      In couples with an oocyte-related OAD, tailoring the superovulation protocol resulted in successful fertilization, term pregnancies, and deliveries. In couples with a sperm-related OAD as determined by PLCζ assay, mouse oocyte activation test, and the assessment of gene mutations and function, AGT was successful. The AGT-revised protocol yielded an even higher fertilization rate than the AGT-initial protocol, resulting in the birth of healthy offspring in all couples who achieved a clinical pregnancy.
      Identificación y tratamiento de hombres con espermatozoides deficientes en fosfolipasa Cz.

      Objetivo

      Identificar y tratar gametos responsables del fallo completo de fecundación con inyección intra-citoplasmática de esperma (ICSI) mediante una nueva propuesta de tratamiento asistido para gametos (AGT).

      Diseño

      Estudio prospectivo de cohortes.

      Lugar

      Centro de medicina reproductiva.

      Pacientes

      Ciento catorce parejas con un numero adecuado de espermatozoides para ICSI y una tasa de fecundación ≤ 10%, tras controlar edad materna.

      Intervenciones

      Parejas con una deficiencia de activación relacionada con el ovocito (OAD) fueron sometidas a un ciclo posterior con un protocolo de superovulación modificado; parejas con OAD relacionada con esperma tuvieron una evaluación genética y epigenética adicional para identificar mutaciones y niveles de expresión de los genes correspondientes.

      Principales medidas de resultado

      Resultados de ciclo para parejas sometidas a ICSI mediante protocolo modificado de superovulación o AGT comparadas con su ciclo previo.

      Resultados

      Un total de 114 parejas cumplieron criterios de inclusión, representando el 1.3% del total de ciclos ICSI realizados en nuestro centro, con controles emparejados de edad. Cincuenta y dos parejas fueron confirmadas como negativas para OAD relacionado con esperma mediante el ensayo de fosfolipasa Cz (PLCz) , indicando factores relacionados con el ovocito para sus ciclos con fallo de fecundación. Las parejas fueron tratadas con uno de los dos protocolos AGT, AGT-inicial o AGT-revisado, en un intento posterior que fue comparado con su ciclo previo. Los ciclos de ICSI posteriores con un protocolo de superovulación personalizado dieron lugar a unas tasas de fecundación (59.0% vs. 2.1%) y gestación clínica (28.6% vs. 0) mayores. En 24 parejas (media ± desviación estándar: edad materna, 35.6 ± 5 años; paterna, 39.8 ±6 años) la OAD relacionada con esperma fue confirmada; en cuatro hombres se identificó una delección relacionada con el gen PLCZ1. Mutaciones adicionales fueron también identificadas en genes relacionados con el mantenimiento de la espermiogenésis y el desarrollo embrionario (PIWIL1, BSX, NLRP5) y delecciones génicas confirmando la ausencia completa de teca perinuclear subacrosomal (PICK1, SPATA16, DPY19L). El posterior tratamiento AGT dio lugar a una mayor tasa de fecundación (42.1%) y gestación clínica (36% vs. 0%) en parejas con historial de fallo de fecundación (9,1%). La comparación de los dos protocolos AGT, AGT-inicial y AGT-revisado, reveló que este ultimo daba lugar a una tasa de fecundación (37.6% vs. 45.9%) y gestación clínica (21.1% vs. 83.3%) aun más favorables.

      Conclusión

      En parejas con OAD relacionada con el ovocito, el protocolo personalizado de superovulación resulto en una fecundación exitosa, gestación a termino y parto. En parejas con una OAD relacionada con el esperma, determinada mediante ensayo PLCz), test de activación de ovocito en raton y evaluación de las mutaciones en genes y función, AGT fue exitoso. El protocolo AGT-revisado dio lugar a tasas aun mayores de fecundación que el protocolo AGT-inicial, dando lugar al nacimiento de bebes sanos en todas las parejas que consiguieron gestación clínica.

      Key Words

      Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/66678-29734
      The implementation of intracytoplasmic sperm injection (ICSI) has greatly alleviated male factor infertility driven by impaired fertilization by overcoming defects of the zona pellucida and sperm acrosomal dysfunction, making it the ultimate assisted reproduction technique for a variety of male infertility indications. These indications can be categorized as either male factor or non–male factor related (
      • Palermo G.D.
      • Neri Q.V.
      • Rosenwaks Z.
      To ICSI or not to ICSI.
      ). Male factor indications encompass poor semen parameters such as oligozoospermia, asthenozoospermia, teratozoospermia, or cryptozoospermia. In addition, ICSI with microsurgically retrieved spermatozoa from the epididymis or testis can be used to treat infertility due to an obstruction of the excretory ducts (
      • Silber S.J.
      • Nagy Z.P.
      • Liu J.
      • Godoy H.
      • Devroey P.
      • Vansteirteghem A.C.
      Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration.
      ,
      • Tournaye H.
      • Devroey P.
      • Liu J.E.
      • Nagy Z.
      • Lissens W.
      • Vansteirteghem A.
      Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection—a new effective approach to infertility as a result of congenital bilateral absence of the vas-deferens.
      ,
      • Mansour R.T.
      • Aboulghar M.A.
      • Serour G.I.
      • Fahmi I.
      • Ramzy A.M.
      • Amin Y.
      Intracytoplasmic sperm injection using microsurgically retrieved epididymal and testicular sperm.
      ). Non–male factor indications for ICSI include cases with prior failed fertilization with in vitro fertilization, oocyte dysmorphism, poor oocyte maturity, or a low number of oocytes (
      • Palermo G.D.
      • Neri Q.V.
      • Rosenwaks Z.
      To ICSI or not to ICSI.
      ). However, although rare, some couples suffer from total fertilization failure with ICSI despite an adequate number of oocytes and spermatozoa.
      Fertilization begins with penetration of the spermatozoon through the zona, followed by fusion with the oolemma where it releases a labile protein, stored in the perinuclear theca, which is responsible for triggering oocyte activation. The compound, identified as phospholipase-Cζ (PLCζ), is delivered into the oocyte after sperm–egg fusion (
      • Swann K.
      • Lai F.A.
      The sperm phospholipase C-zeta and Ca2+ signalling at fertilization in mammals.
      ). At the same time, cytosolic calcium ion (Ca2+) oscillations characterized by a series of distinct Ca2+ spikes take place within the oocyte, eventually leading to successful fertilization (
      • Wolny Y.M.
      • Fissore R.A.
      • Wu H.
      • Reis M.M.
      • Colombero L.T.
      • Ergun B.
      • et al.
      Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes.
      ,
      • Tavalaee M.
      • Nomikos M.
      • Lai F.A.
      • Nasr-Esfahani M.H.
      Expression of sperm PLCζ and clinical outcomes of ICSI-AOA in men affected by globozoospermia due to DPY19L2 deletion.
      ). At gamete fusion, the sperm aster is formed by the centriolar region while sperm nuclear decondensation takes place. The aster then guides the female pronucleus toward the male pronucleus, forming the first mitotic spindle, while the centriole duplicates during the pronuclear stage, culminating in syngamy (
      • Palermo G.D.
      • Colombero L.T.
      • Rosenwaks Z.
      The human sperm centrosome is responsible for normal syngamy and early embryonic development.
      ,
      • Sathananthan A.H.
      • Ratnam S.S.
      • Ng S.C.
      • Tarin J.J.
      • Gianaroli L.
      • Trounson A.
      The sperm centriole: its inheritance, replication and perpetuation in early human embryos.
      ).
      Oocyte activation that precedes fertilization can only occur in a receptive ooplasm (
      • Whitaker M.
      Calcium at fertilization and in early development.
      ), so poor ICSI fertilization outcomes may also be due to oocyte-related factors, involving ooplasmic dysmaturity (
      • Pereira N.
      • Neri Q.V.
      • Lekovich J.P.
      • Palermo G.D.
      • Rosenwaks Z.
      The role of in-vivo and in-vitro maturation time on ooplasmic dysmaturity.
      ). The source of fertilization failure being due to male gamete defects is more readily accepted, and oocyte-related factors are often overlooked. Indeed, a study investigating the effects of the timing of in vivo and in vitro maturation on ooplasmic dysmaturity concluded that modulating the time intervals between the human chorionic gonadotropin (hCG) trigger, oocyte retrieval, removal of cumulus cells, and ICSI execution can improve the likelihood of fertilization in couples with a history of complete fertilization failure in the presence of apparently normal gametes (
      • Pereira N.
      • Neri Q.V.
      • Lekovich J.P.
      • Palermo G.D.
      • Rosenwaks Z.
      The role of in-vivo and in-vitro maturation time on ooplasmic dysmaturity.
      ). Specifically, adjusting the ovarian stimulation protocol to allow for longer in vivo and in vitro maturation in the presence of cumulus/corona cells enhances fertilization rates.
      Superovulation protocols aimed at achieving a higher proportion of metaphase 2 (MII) oocytes would also be effective in achieving better fertilization and pregnancy outcomes (
      • Parrella A.
      • Irani M.
      • Keating D.
      • Chow S.
      • Rosenwaks Z.
      • Palermo G.D.
      High proportion of immature oocytes in a cohort reduces fertilization, embryo development, pregnancy and live birth rates following ICSI.
      ). It has also been shown that fertilization rates and embryo developmental competence of inseminated oocytes are directly related not only to nuclear but to ooplasmic and membrane maturity (
      • Shu Y.
      • Gebhardt J.
      • Watt J.
      • Lyon J.
      • Dasig D.
      • Behr B.
      Fertilization, embryo development, and clinical outcome of immature oocytes from stimulated intracytoplasmic sperm injection cycles.
      ,
      • Yanez L.Z.
      • Han J.
      • Behr B.B.
      • Pera R.A.R.
      • Camarillo D.B.
      Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization.
      ), indicating that for fertilization to occur, oocytes must have a responsive mature cytoplasm capable of generating proper Ca2+ oscillation patterns after sperm exposure.
      Nonetheless, impaired fertilization is often attributable to the male gamete. It can be due to complete spermatozoa immotility or teratozoospermia associated with abnormalities of the sperm head or the flagellum, all of which may affect transport of the sperm and/or acrosomal function, respectively (
      • Flaherty S.P.
      • Payne D.
      • Swann N.J.
      • Mattews C.D.
      Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection.
      ). To correct sperm-related fertilization failure, a variety of assisted oocyte activation (AOA) methods have been proposed aimed at triggering the release of intracellular Ca2+, generating calcium spikes followed by the initiation of meiosis (
      • Bonte D.
      • Ferrer-Buitrago M.
      • Dhaenens L.
      • Popovic M.
      • Thys V.
      • De Croo I.
      • et al.
      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 there have been many studies supporting the effectiveness of AOA treatment (
      • Vanden Meerschaut F.
      • Nikiforaki D.
      • Heindryckx B.
      • De Sutter P.
      Assisted oocyte activation following ICSI fertilization failure.
      ), most have failed to identify gamete-specific causes. Indeed, a review of the literature revealed that out of 55 studies, only 11 assessed for sperm-related OAD before using AOA (
      • Rubino P.
      • Vigano P.
      • Luddi A.
      • Piomboni P.
      The ICSI procedure from past to future: a systematic review of the more controversial aspects.
      ). The importance of performing such an assessment was highlighted by a report categorizing patients according to the results of the mouse oocyte activation test (MOAT) in relation to AOA outcome, concluding that AOA is best reserved for cases with a clear sperm-related OAD (
      • Bonte D.
      • Ferrer-Buitrago M.
      • Dhaenens L.
      • Popovic M.
      • Thys V.
      • De Croo I.
      • et al.
      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.
      ).
      However, MOAT grading has a wide span of interpretations that can be equivocal in nature, and an argument could be made that it is not an ideal diagnostic test to assess the underlying cause of fertilization failure. Because the test itself is heterospecific, the injection of human spermatozoa into mouse oocytes may not provide, by itself, the most accurate results. It is also cumbersome and time consuming, and can only be performed in a setting with access to an animal facility where mouse oocytes may be stimulated and harvested (
      • Neri Q.V.
      • Lee B.
      • Rosenwaks Z.
      • Machaca K.
      • Palermo G.D.
      Understanding fertilization through intracytoplasmic sperm injection (ICSI).
      ). Therefore, a direct PLCζ assessment by immunofluorescence appears to be a feasible and more reliable technique to evaluate the oocyte-activating capacity of spermatozoa.
      The diagnosis of a clear sperm-related OAD should be made before considering AOA, especially since an isolated absence of PLCζ is rare. Indeed, its uncommon prevalence, together with the lack of sperm cytosolic factor assessment (
      • Ebner T.
      • Montag M.
      • Montag M.
      • Van der Ven K.
      • Van der Ven H.
      • Ebner T.
      • et al.
      Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.
      ,
      • Montag M.
      • Koster M.
      • van der Ven K.
      • Bohlen U.
      • van der Ven H.
      The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle.
      ), would imply that, contrary to what has been proposed by the recent literature, most cases with fertilization failure are not solely related to the male gamete and may not actually require AOA treatment. Therefore, it is crucial to determine whether AOA is truly necessary or if successful fertilization can be achieved by optimizing ooplasmic maturity.
      In this study, we specifically allocated couples with complete failed fertilization after ICSI by the gamete responsible. To identify these couples, we screened the spermatozoa with a PLCζ assay, confirmed by MOAT, and corroborated our findings by genomic assessments. Only in couples with a confirmed sperm-related OAD did we apply assisted gamete treatment (AGT) in subsequent ICSI cycles.

      Materials and methods

       Inclusion Criteria and Study Design

      Over a 20-year period, 114 out of 9,326 couples undergoing treatment for infertility at our center were included in our study. These couples presented with a history of either extremely poor (≤10%) or complete fertilization failure after undergoing ICSI cycles in which ≥3 oocytes were inseminated. The minimum number of three oocytes was chosen to include a larger number of patients who met the threshold while still allowing us to exclude ICSI technical error. This population included only couples where the male partner had a sperm concentration of ≥1 × 106/mL and whose female partner was ≤37 years of age and had a normal infertility workup. The components of an infertility evaluation for the female partners consisted of a comprehensive review of the medical history as well as a targeted physical examination and additional tests focusing on ovarian reserve, ovulatory function, and uterine structural abnormalities. Patients with a normal workup were not found to have any abnormalities in these assessments.
      Thirty-eight of these couples elected to withhold or continue infertility treatment elsewhere, but for those couples continuing treatment at our center (n = 76), OAD was assessed to detect the presence of sperm cytosolic factor in the perinuclear theca of the sperm head. Couples with a negative PLCζ assay (n = 52), where OAD was considered oocyte-related, underwent subsequent ICSI cycles with a tailored stimulation protocol in which the time intervals between hCG trigger, oocyte retrieval, denudation, and ICSI were modified (
      • Pereira N.
      • Neri Q.V.
      • Lekovich J.P.
      • Palermo G.D.
      • Rosenwaks Z.
      The role of in-vivo and in-vitro maturation time on ooplasmic dysmaturity.
      ,
      • Elias R.T.
      • Pereira N.
      • Palermo G.D.
      The benefits of dual and double ovulatory triggers in assisted reproduction.
      ).
      On the other hand, when the screening assay was positive and the poor fertilization was presumed to be caused by a sperm-related OAD (n = 24), the couples underwent ensuing treatment with AGT. After the PLCζ assay and confirmatory MOAT, the consenting men (n = 4) from this cohort were also evaluated with a genetic assessment by DNAseq and an epigenetic assessment using RNAseq to identify mutations and expression levels of related genes.
      In all couples, once the underlying etiology of the fertilization failure (female or male gamete) was determined and the appropriate treatment cycles were performed, the clinical outcomes in terms of fertilization, pregnancy, and delivery were compared with the same couples’ historical cycles before treatment. In a subanalysis, we also compared the effectiveness of two different AGT protocols in which spermatozoa and postinjection oocytes were exposed to either streptolysin O or calcium ionophore. A decision tree flowchart has been included to clarify our screening process and treatment approach (Supplemental Fig. 1, available online).
      This study was approved by the institutional review board of New York Presbyterian Hospital-Weill Cornell Medicine (IRB 0712009553). All patients gave informed written consent to participate.

       Spermatozoa Collection and Preparation

      Ejaculates were provided by masturbation and were evaluated according to World Health Organization standards (
      • World Health Organization
      Laboratory manual for the examination and processing of human semen.
      ). Specimens were centrifuged after 3:1 dilution in human tubal fluid medium (Irvine Scientific) supplemented with human serum albumin (HSA-Solution; Vitrolife) at 600 × g for 10 minutes to remove the seminal fluid. In preparation for PLCζ assessment, specimens were smeared on a glass slide and allowed to dry overnight. For MOAT, a final suspension of the sperm specimen of approximately 3 × 106/mL concentration was prepared. For DNA and RNA sequencing, the sperm concentration was adjusted to 500 cells/mL and 4–5 × 106/mL, respectively.

       PLCζ Staining and Assessment

      For PLCζ assessment, 10 μL of sperm specimen was smeared on slides and fixed in 4% paraformaldehyde. The slides were then permeabilized in 0.5% Triton X-100/phosphate-buffered saline for 5 minutes on ice, then incubated overnight with polyclonal anti-PLCζ antibody. The specimen was labeled with a secondary antibody, anti-rabbit IgG, and counterstained with 5 μg/mL of 4ʹ,6-diamidino-2-phenylindole (DAPI). Slides were imaged at ×100 original magnification on a fluorescence microscope using a brightfield filter and a fluorescein isothiocyanate (FITC) filter (Supplemental Fig. 2, available online). The percentage of spermatozoa exhibiting PLCζ immunofluorescence in the acrosomal, equatorial, and postacrosomal regions of the head was recorded for at least 200 cells per specimen. By assessing a large number of patients for PLCζ, we found that those with less than 30% PLCζ yielded consistently low or unobtainable fertilization. Therefore, a normal threshold of ≥30% was used.

       Mouse Oocyte Activation Test

      Mouse oocytes were retrieved from B6D2 F1 hybrid female mice after ovulation induction with 0.2 mL of pregnant mare’s serum gonadotropin (PMSG) and inhibin cocktail (CARD Hyperova; Cosmo Bio). Approximately 46–48 hours later, 7.5 IU hCG (Chorulon; Intervet) was administered. Cumulus–oocyte complexes were then collected from the oviducts 14–16 hours after hCG administration. Culture and handling media were potassium simplex optimized medium (KSOM; Cosmo Bio) and M2-HEPES (M2; Cosmo Bio), respectively. Cumulus cells were removed by briefly exposing the oocytes to 80 IU hyaluronidase/mL (EmbryoMax M2 medium with hyaluronidase; Sigma-Aldrich). Piezo-actuated ICSI using patient spermatozoa was performed at 15–17°C in M2-HEPES, supplemented with 20% fetal bovine serum (Gibco PBRL). First, several piezo pulses were applied to perforate the zona pellucida. Next, the spermatozoon was moved to the tip of the micropipette, which was traversed into the oocyte. A gentle piezo pulse was then applied to break the oolemma and allow the spermatozoon to be deposited into the cytoplasm (
      • Yoshida N.
      • Perry A.C.
      Piezo-actuated mouse intracytoplasmic sperm injection (ICSI).
      ).
      Positive and negative controls were established by injecting donor spermatozoa with proven fertility or sham ICSI, respectively. Noninjected oocytes were observed to exclude spontaneous parthenogenetic activation. All oocytes were kept in culture for 1 day, and the percentage of successful oocyte activation was determined by examining the number of oocytes with two polar bodies and two pronuclei, as well as their development to the two-cell stage (
      • Egashira A.
      • Murakami M.
      • Haigo K.
      • Horiuchi T.
      • Kuramoto T.
      A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation.
      ,
      • Yazawa H.
      • Yanagida K.
      • Katayose H.
      • Hayashi S.
      • Sato A.
      Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.
      ). A threshold of ≥20% was considered normal. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of New York Presbyterian Hospital-Weill Cornell Medicine (0605-493A).

       Genetic and Epigenetic Assessment

      To identify gene mutations related to the spermatozoal oocyte-activating potential, we performed DNA sequencing as described elsewhere (
      • Cheung S.
      • Schlegel P.N.
      • Rosenwaks Z.
      • Palermo G.D.
      Revisiting aneuploidy profile of surgically retrieved spermatozoa by whole exome sequencing molecular karyotype.
      ). The DNA extraction and amplification were achieved with a commercial kit (Repli-G Single Cell; Qiagen), yielding a concentration of 1,487.7 ± 264 ng/μL and quality of 1.7 ± 0.1. Specimens were then sequenced at an external facility (Genewiz). Genes were considered duplicated when the read depth was greater than 1.5 times the median depth, or number of unique reads, in the control group for >70% exons in the gene and were considered deleted when the read depth was less than 0.5 times the median depth in the control.
      To assess the expression levels of genes related to spermatozoa oocyte-activating potential, RNA sequencing was performed as described elsewhere (
      • Cheung S.
      • Parrella A.
      • Rosenwaks Z.
      • Palermo G.D.
      Genetic and epigenetic profiling of the infertile male.
      ). Purified total RNA was obtained from lysed spermatozoa using a hybrid protocol with TRIzol Reagent (ThermoFisher Scientific) and a RNeasy Mini Kit spin column (RNEasy; Qiagen). To confirm the quality of potential reads, a pilot paired-end 36 base pair (bp) RNA-sequencing was run (NextSeq 500; Illumina) by an external laboratory (Genewiz), and then expanded to 50–60 M reads at 2×75 bp. Sequenced reads were mapped to the hg20 reference genome; for differential expression analysis, an algorithm by edgeR and CONTRA (LGPL; Bioconductor) was implemented to assess fragments per kilobase of transcript per million mapped reads.

       Ovarian Superovulation and Oocyte Collection

      To determine the stimulation protocol, the patient age, weight, antral follicular count, serum antimüllerian hormone level, and previous response to stimulation were carefully examined (
      • Huang J.Y.
      • Rosenwaks Z.
      Assisted reproductive techniques.
      ). Patients were treated with daily gonadotropins (Follistim; Merck; Gonal-F, EMD-Serono; and/or Menopur, Ferring Pharmaceuticals), and pituitary suppression was achieved by gonadotropin-releasing hormone (GnRH) agonist (leuprolide acetate, Abbott Laboratories) or GnRH antagonist (Ganirelix acetate, Merck; or Cetrotide, EMD-Serono). To attain follicular synchronization, some patients were treated with oral contraceptive pills (Ortho-Novum, Janssen Pharmaceuticals) before starting gonadotropins. The hCG trigger (Ovidreal, EMD-Serono) for final oocyte maturation was administered when at least two lead follicles reached an average diameter of ≥17 mm. Transvaginal oocyte retrieval was performed under conscious sedation 35–37 hours after hCG administration.
      The oocytes were further incubated for an additional 3 to 4 hours after retrieval. Before micromanipulation, the cumulus–corona cells were removed by exposing the oocytes to medium containing 40 IU/mL of hyaluronidase (Cumulase; Halozyme Therapeutics) (
      • Neri Q.V.
      • Lee B.
      • Rosenwaks Z.
      • Machaca K.
      • Palermo G.D.
      Understanding fertilization through intracytoplasmic sperm injection (ICSI).
      ,
      • Palermo G.D.
      • Cohen J.
      • Alikani M.
      • Adler A.
      • Rosenwaks Z.
      Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility.
      ). To facilitate this process, oocytes were aspirated in and out of a calibrated pipette stripper (Origio) with an approximately 200-μm inner diameter. Complete removal of the adhering corona radiata was necessary to prevent visual obstruction of the oocyte as well as the holding and/or injection pipettes by the residual corona cells.
      Each oocyte was washed twice in culture medium (home-brew, modified Cornell medium based on G1 and G2 components; Vitrolife) (
      • Gardner D.K.
      • Lane M.
      • Spitzer A.
      • Batt P.A.
      Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development.
      ,
      • Gardner D.K.
      • Lane M.
      Culture and selection of viable blastocysts: a feasible proposition for human IVF?.
      ) and then examined under the inverted microscope (TE2000U; Nikon USA) equipped with ×2, ×4, and ×10 objectives (Nikon CFI Apo), and ×20 and ×40 objectives (Nikon Polarized optics CFI Plan Fluor) to assess integrity and maturational stage. After cumulus removal, oocytes at prophase I displayed a germinal vesicle, and at metaphase I, the germinal vesicle breaks down without extrusion of the polar body. Once the first polar body was identified, oocytes were considered to be at the MII stage and ready for ICSI. For couples with a history of fertilization failure, adjustment of the stimulation protocol was performed according to our prior experience to improve ooplasmic maturity and increase the proportion of MII oocytes recovered (
      • Parrella A.
      • Irani M.
      • Keating D.
      • Chow S.
      • Rosenwaks Z.
      • Palermo G.D.
      High proportion of immature oocytes in a cohort reduces fertilization, embryo development, pregnancy and live birth rates following ICSI.
      ,
      • Elias R.T.
      • Pereira N.
      • Palermo G.D.
      The benefits of dual and double ovulatory triggers in assisted reproduction.
      ).

       ICSI with Assisted Gamete Treatment

      We performed ICSI in conjunction with one of two activation protocols: AGT-initial or AGT-revised (Fig. 1). For the AGT-initial protocol, spermatozoa were exposed to streptolysin O (SLO), a streptococcal secretory protein that permeabilizes cells by interacting with cholesterol in the plasma membrane, for 30 minutes before spermatozoa injection (
      • Neri Q.V.
      • Lee B.
      • Rosenwaks Z.
      • Machaca K.
      • Palermo G.D.
      Understanding fertilization through intracytoplasmic sperm injection (ICSI).
      ). The details of the injection procedure have been described elsewhere, as have the selection of the spermatozoon and the immobilization-permeabilization method (
      • Palermo G.D.
      • Cohen J.
      • Alikani M.
      • Adler A.
      • Rosenwaks Z.
      Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility.
      ). Oocytes were incubated in 10 μM calcium ionophore (Sigma-Aldrich) for two rounds of 10 minutes at 37°C, 30 minutes after ICSI injection, and then were rinsed and transferred into C1 culture medium.
      Figure thumbnail gr1
      Figure 1Assisted gamete treatment (AGT) intracytoplasmic sperm injection (ICSI) mechanism. Couples with a confirmed sperm-related oocyte activation deficiency (OAD) underwent subsequent ICSI cycles with either the AGT-initial or AGT-revised protocol. (A) For AGT-initial, spermatozoa were briefly exposed to streptolysin O (SLO) before ICSI injection. Postinjection oocytes were then incubated in calcium ionophore for two rounds, then rinsed and transferred to culture medium. (B) For AGT-revised, spermatozoa were briefly exposed to calcium ionophore before ICSI. After injection, oocytes were incubated in calcium ionophore before being transferred to the culture medium.
      For the AGT-revised protocol, ejaculated spermatozoa were briefly exposed to calcium ionophore in a drop on the ICSI dish before injection. During the ICSI procedure, spermatozoa were aspirated individually from the drop containing calcium ionophore and immobilized in a separate polyvinylpyrrolidone drop. Next, approximately 0.4 pL of calcium ionophore was aspirated into the micropipette and injected into the oocyte with the spermatozoa. Post-ICSI oocytes were then exposed to 50 μM calcium ionophore for 10 minutes at 37°C, and then washed and placed in culture medium.

       Fertilization Assessment, Embryo Transfer, Pregnancy Assessment, and Delivery Follow-up

      The assessment of successful activation/fertilization was performed under an inverted microscope 16–18 hours after ICSI by evaluating oocytes for the presence of two clear pronuclei and two distinct polar bodies (
      • Palermo G.D.
      • Cohen J.
      • Alikani M.
      • Adler A.
      • Rosenwaks Z.
      Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility.
      ). The oocytes were then loaded into the EmbryoScope incubator (EmbryoScope; Vitrolife) to be monitored by time-lapse imaging thereafter.
      Embryo transfer was performed 3 or 5 days after microinjection. Patients received 50 mg of intramuscular progesterone supplement daily, starting 24 hours after retrieval. Serum β-hCG levels were then measured 14 days after retrieval. Clinical pregnancy was defined as fetal heart activity detected on ultrasound at 7 weeks’ gestation. Information on delivery method and neonatal conditions were obtained through the obstetric and pediatric records.

       Statistical Analysis

      Friedman’s chi-square and Fisher’s exact tests (Jandel Scientific) were used to compare the outcomes generated between conventional ICSI cycles and subsequent cycles with a modified stimulation protocol or AGT. P<.05 was considered statistically significant. A post hoc power analysis was also performed using the McNemar’s test for data presented in Tables 2 and 3.

      Results

      A total of 114 couples (maternal age, 33.8 ± 4 years; paternal age, 36.9 ± 5 years) with a history of poor or complete failed fertilization (≤10%) were included (Supplemental Fig. 1). The patients’ ages and semen parameters are presented in Table 1. Although 38 of these couples elected to withhold infertility treatment or to be treated elsewhere, the remaining 76 opted to undergo subsequent ICSI cycles at our center. These couples were screened by PLCζ assay to determine whether poor fertilization was attributed to a sperm-related OAD or to ooplasmic dysmaturity (oocyte-related OAD).
      Table 1Couples’ demographic and gamete characteristics, and intracytoplasmic sperm injection outcome.
      CharacteristicAll couples
      Couples, n114
       Female age (y)33.8 ± 4
       Male age (y)36.9 ± 5
      Semen parameter
       Concentration, 106/mL ± SD49.2 ± 51
       Motility, % ± SD43 ± 19
       Morphology, % ± SD2.9 ± 2
      ICSI cycles, n119
       Oocytes retrieved, n1,145
       MII, n (%)851 (74.3)
       2PN, n (%)38 (4.4)
       Cycles with ET20
       Clinical pregnancy per cycle, n (%)0 (0)
       Clinical pregnancy (+FHB), n (%)0 (0)
      Note: +FHB = presence of at least one fetal heartbeat; 2PN = two pronuclei; ET = embryo transfer; ICSI = intracytoplasmic sperm injection; MII = metaphase 2; SD = standard deviation.
      The PLCζ assay was negative on the spermatozoa of 52 couples, indicating that the cause of fertilization failure of their cycles was likely due to ooplasmic dysmaturity. In previous cycles, an average of 6.4 oocytes were injected with motile spermatozoa with an observed fertilization rate of only 2.1% (7 of 334). Of these, six couples underwent embryo replacement, but none achieved a clinical pregnancy. Thus, they consented to be treated in subsequent ICSI cycles with a modified stimulation protocol. An average of 7.6 oocytes were injected with motile spermatozoa in 105 ICSI cycles, yielding an improved fertilization rate of 59% (470 of 796; P<.0001). Ninety-one cycles resulted in embryo replacement, yielding 30 clinical pregnancies (28.6%; P<.0001) with five pregnancy losses and 25 deliveries (Table 2). A total of 32 babies were born, 15 boys and 17 girls, with no major or minor congenital malformations.
      Table 2Intracytoplasmic sperm injection outcome for couples with confirmed oocyte- or sperm-related oocyte activation deficiency.
      CharacteristicTotalControlModified superovulation protocol
      Oocyte-related OAD
       Couples, n52
      Female age (y)33.1 ± 433.4 ± 3
      Male age (y)35.7 ± 535.7 ± 5
       ICSI cycles, n52105
      Oocytes retrieved, n4561,120
      MII, n (%)334 (73.2)796 (71.1)
      2PN, n (%)7 (2.1)
      Chi-square, 2×2, 1 df, P<.0001.
      470 (59.0)
      Chi-square, 2×2, 1 df, P<.0001.
      Cycles with ET, n691
      Clinical pregnancy per cycle, n (%)0 (0)
      Chi-square, 2×2, 1 df, P<.0001.
      30 (28.6)
      Chi-square, 2×2, 1 df, P<.0001.
      Clinical pregnancy (+FHB), n (%)0 (0)30 (32.9)
      Deliveries25 (83.3)
      TotalControlAssisted gamete treatment
      Sperm-related OAD
       Couples, n24
      Female age (y)35.6 ± 5
      Male age (y)39.8 ± 6
       ICSI cycles, n2743
      Oocytes retrieved, n230404
      MII, n (%)197 (85.7)323 (79.9)
      2PN, n (%)18 (9.1)
      Chi-square, 2×2, 1 df, P<.05.
      136 (42.1)
      Chi-square, 2×2, 1 df, P<.05.
      Cycles with ET, n425
      Clinical pregnancy per cycle, n (%)0 (0)
      Chi-square, 2×2, 1 df, P<.05.
      9 (20.9)
      Chi-square, 2×2, 1 df, P<.05.
      Clinical pregnancy (+FHB), n (%)0 (0)
      Chi-square, 2×2, 1 df, P<.05.
      9 (36.0)
      Chi-square, 2×2, 1 df, P<.05.
      Deliveries, n6
      Note: Data presented as mean ± standard deviation, unless noted otherwise. +FHB = presence of at least one fetal heartbeat; 2PN = two pronuclei;.df = degrees of freedom; ET = embryo transfer;ICSI = intracytoplasmic sperm injection; MII = metaphase 2; OAD = oocyte activation deficiency.
      a,b Chi-square, 2×2, 1 df, P<.0001.
      c,d,e Chi-square, 2×2, 1 df, P<.05.
      Sperm cytosolic factor assessment resulted in a positive PLCζ assay on the spermatozoa of 24 patients. We performed MOAT to validate these findings and confirmed that the spermatozoa displayed dysfunctional oocyte-activating capacity.
      The DNA sequencing on spermatozoa from patients with sperm-related OAD revealed mutations on several genes. Most importantly, the men (n = 4) analyzed had a deletion on the PLCZ1 gene, confirming the absence of PLCζ and corroborating the findings of our screening assays. Additionally, genes involved in supporting spermiogenesis and embryo development (PIWIL1, BSX, NLRP5) were identified. Deletions were also detected for PICK1, SPATA16, and DPY19L, confirming a complete absence of the subacrosomal perinuclear theca, a finding also observed in globozoospermic men. RNAseq confirmed the expression imbalance of the abovementioned genes.
      These results allowed us to effectively identify cycles in which fertilization failure was caused by a sperm-related OAD. In 27 ICSI cycles performed without AGT, an average of 7.3 oocytes were injected with motile spermatozoa, yielding a fertilization rate of 9.1% (18 of 197). However, although four couples underwent embryo transfers, none achieved a clinical pregnancy. When these patients underwent subsequent ICSI cycles with AGT, their fertilization and clinical pregnancy rates significantly increased to 42.1% (136 of 323; P<.05) and 36.0% (9 of 25; P<.05), respectively. Six patients have successfully delivered (Table 2), and the offspring are displaying normal development at 3 years of age.
      In the 24 couples whose poor fertilization cycles were confirmed to be caused by a sperm-related OAD, ICSI using either an initial-AGT protocol, in which the spermatozoa were treated with SLO, or a revised-AGT protocol, where both gametes were exposed solely to calcium ionophore, was performed. When outcomes between the 24 AGT-initial and 18 AGT-revised cycles were compared, showing a fertilization rate trending upward from 37.6% to 45.9%, both approaches resulted in more favorable outcomes than in the index cycles performed without either of the AGT protocols (P<.05). Although there was no notable difference in fertilization rates between the AGT-initial and AGT-revised cycles, the treatment of gametes with the AGT-revised protocol using only calcium ionophore resulted in a significantly higher clinical pregnancy rate than the cycles treated with the AGT-initial technique (83.3% vs. 21.1%; P<.05) (Table 3).
      Table 3Comparison of initial and revised assisted gamete treatment protocols.
      CharacteristicControlAGT
      InitialRevised
      Couples, n241113
       Female age (y)35.6 ± 536.3 ± 332.3 ± 6
       Male age (y)39.8 ± 639.6 ± 335.8 ± 6
      ICSI cycles, n272418
       Oocytes retrieved, n230181223
       MII, n (%)197 (85.7)149 (82.3)174 (78.1)
       2PN, n (%)18 (9.1)
      Chi-square, 2×2, 1 df, P<.05.
      ,
      Chi-square, 2×2, 1 df, P<.05.
      56 (37.6)
      Chi-square, 2×2, 1 df, P<.05.
      80 (45.9)
      Chi-square, 2×2, 1 df, P<.05.
       Cycles with ET, n4196
       Clinical pregnancy per cycle, n (%)0 (0)
      Chi-square, 2×2, 1 df, P<.05.
      ,
      Chi-square, 2×2, 1 df, P<.05.
      4 (16.7)
      Chi-square, 2×2, 1 df, P<.05.
      5 (27.8)
      Chi-square, 2×2, 1 df, P<.05.
       Clinical pregnancy (+FHB), n (%)0 (0)
      Chi-square, 2×2, 1 df, P<.05.
      4 (21.1)
      Chi-square, 2×2, 1 df, P<.05.
      5 (83.3)
      Chi-square, 2×2, 1 df, P<.05.
      ,
      Chi-square, 2×2, 1 df, P<.05.
       Deliveries15
      Note: Data presented as mean ± standard deviation, unless noted otherwise. +FHB = presence of at least one fetal heartbeat; 2PN = two pronuclei;.AGT= assisted gamete treatment; df = degrees of freedom; ET = embryo transfer; ICSI = intracytoplasmic sperm injection; MII = metaphase 2.
      a,b,c,d,e,f Chi-square, 2×2, 1 df, P<.05.
      Furthermore, AGT-initial cycles resulted in the delivery of one healthy baby boy whereas AGT-revised cycles resulted in the births of seven healthy offspring—three singletons and two sets of twins. There were no major or minor congenital malformations in the resulting five girls and two boys, all of whom are developing well with no notable developmental delays at 3 years of age.

      Discussion

      To our knowledge, ours is the first study that attempts to attribute ICSI fertilization failure to a specific gamete. Although there is a substantial body of literature supporting the use of oocyte-activation treatments, very few studies have demonstrated fertilization failure that can be ascribed to the male gamete. Indeed, one report on 89 couples found that the use of calcium ionophore significantly increased fertilization rates compared with those of previous cycles (
      • Montag M.
      • Koster M.
      • van der Ven K.
      • Bohlen U.
      • van der Ven H.
      The benefit of artificial oocyte activation is dependent on the fertilization rate in a previous treatment cycle.
      ). However, only 27 (30%) of these couples presented with a history of complete fertilization failure, and the remaining couples had a history of fertilization up to 50% with conventional ICSI. The study also neglects to assess for a sperm cytosolic factor to confirm whether all cases of fertilization failure were truly due to a sperm-related OAD. Another report on 101 couples also evidenced a significantly higher fertilization with AOA than with conventional ICSI (
      • Ebner T.
      • Montag M.
      • Montag M.
      • Van der Ven K.
      • Van der Ven H.
      • Ebner T.
      • et al.
      Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.
      ). But again, although it accrued a large study population, only a small fraction (n = 15; 14.9%) actually had a history of complete fertilization failure with conventional ICSI. These are not isolated reports; indeed, in a comprehensive literature assessment of 47 AOA studies (
      • Rubino P.
      • Vigano P.
      • Luddi A.
      • Piomboni P.
      The ICSI procedure from past to future: a systematic review of the more controversial aspects.
      ), only two screened for a sperm-related OAD (
      • Rubino P.
      • Vigano P.
      • Luddi A.
      • Piomboni P.
      The ICSI procedure from past to future: a systematic review of the more controversial aspects.
      ,
      • Rybouchkin A.V.
      • Van der Straeten F.
      • Quatacker J.
      • De Sutter P.
      • Dhont M.
      Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity.
      ,
      • Kyono K.
      • Nakajo Y.
      • Nishinaka C.
      • Hattori H.
      • Kyoya T.
      • Ishikawa T.
      • et al.
      A birth from the transfer of a single vitrified-warmed blastocyst using intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient.
      ).
      In our study, we screened all semen specimens of couples presenting with complete or severe fertilization failure. With this approach, we were able to identify couples with fertilization failure due to oocyte-related OAD, which can be attributed to ooplasmic dysmaturity. A simple modulation of the superovulation protocol performed in a subsequent ICSI cycle was able to successfully improve the fertilization rate (59.0%; P<.0001). In an earlier study, which included 125 patients with a history of failed fertilization, an enhanced clinical outcome was attributed to an increased proportion of MII oocytes in subsequent cycles (
      • Kahyaoglu I.
      • Demir B.
      • Turkkani A.
      • Cinar O.
      • Dilbaz S.
      • Dilbaz B.
      • et al.
      Total fertilization failure: is it the end of the story?.
      ). In a groundbreaking study, we were able to show the relevance of ooplasmic readiness in couples who had complete fertilization failure with ICSI, despite the demonstration of normal male gametes. In this study, an increased in vitro/in vivo maturation time was able to provide oocytes with comparable nuclear maturity but a more receptive cytoplasm (
      • Pereira N.
      • Neri Q.V.
      • Lekovich J.P.
      • Palermo G.D.
      • Rosenwaks Z.
      The role of in-vivo and in-vitro maturation time on ooplasmic dysmaturity.
      ). This was further validated by a later report, which showed higher fertilization and clinical pregnancy rates when a tailored stimulation protocol with a dual ovulatory trigger was implemented (
      • Elias R.T.
      • Pereira N.
      • Palermo G.D.
      The benefits of dual and double ovulatory triggers in assisted reproduction.
      ). Finally, in a large retrospective cohort, over 7,000 cycles were allocated to four groups with a different proportion of immature oocytes at retrieval. The cohort with a low or “minimal” proportion of MII oocytes had the lowest fertilization, implantation, and live-birth rates. This suggested that despite the extrusion of the first polar body, an MII oocyte may, in fact, lack embryo developmental competence (
      • Parrella A.
      • Irani M.
      • Keating D.
      • Chow S.
      • Rosenwaks Z.
      • Palermo G.D.
      High proportion of immature oocytes in a cohort reduces fertilization, embryo development, pregnancy and live birth rates following ICSI.
      ).
      In a recent study in which the gamete competence was assessed by MOAT, a wide array of results was reported, allowing the classification of couples according to their different degrees of OAD. Only the most severe cases of OAD had the highest response to oocyte activation, while for the other categories an oocyte contribution for failed fertilization could not be excluded (
      • Bonte D.
      • Ferrer-Buitrago M.
      • Dhaenens L.
      • Popovic M.
      • Thys V.
      • De Croo I.
      • et al.
      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.
      ). MOAT, however, entails the injection of human spermatozoa into mouse oocytes; therefore, due to its heterospecific nature, it may be inconsistent, inaccurate, or inconclusive. Additionally, the test requires an animal facility where mice undergo ovulatory stimulation and oocyte harvest (
      • Neri Q.V.
      • Lee B.
      • Rosenwaks Z.
      • Machaca K.
      • Palermo G.D.
      Understanding fertilization through intracytoplasmic sperm injection (ICSI).
      ,
      • Cheung S.
      • Xie P.
      • Palermo G.D.
      When to jump-start fertilization.
      ).
      To obviate the aforementioned limitations, we screened for the presence of sperm-related OAD with a more specific assay—the immunofluorescent staining of PLCζ—that allowed us to identify an impaired sperm fertilizing competence (
      • Swann K.
      • Lai F.A.
      The sperm phospholipase C-zeta and Ca2+ signalling at fertilization in mammals.
      ,
      • Nomikos M.
      • Yu Y.
      • Elgmati K.
      • Theodoridou M.
      • Campbell K.
      • Vassilakopoulou V.
      • et al.
      Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility.
      ). To corroborate our findings, we also used MOAT to confirm the absence of sperm cytosolic activating factor. In addition, we performed genomic analyses by next-generation sequencing on the male gamete to identify possible inheritable genetic traits. This would optimize the identification of gene mutations in the germline that may be missed in somatic cell assessments (
      • Berchuck A.
      • Heron K.A.
      • Carney M.E.
      • Lancaster J.M.
      • Fraser E.G.
      • Vinson V.L.
      • et al.
      Frequency of germline and somatic BRCA1 mutations in ovarian cancer.
      ). In those men tested, we confirmed a mutation of the PLCZ1 gene, suggesting a genetic etiology for their inability to fertilize oocytes. A separate genomic analysis confirmed our findings by demonstrating that the absence of PLCZ1 prevents oocyte activation (
      • Escoffier J.
      • Lee H.C.
      • Yassine S.
      • Zouari R.
      • Martinez G.
      • Karaouzene T.
      • et al.
      Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP.
      ). We also found that several genes involved in acrosomal development, sperm–egg fusion, and embryo development were mutated. Furthermore, these results were solidified by gene function profiling through RNAseq.
      The clear identification of a sperm-related OAD prompted us to treat these couples in subsequent ICSI cycles by AGT, allowing them to achieve higher fertilization (42.1%; P<.05) and pregnancy rates (20.9%; P<.05). A similar outcome was achieved by treating both gametes, resulting in increased fertilization rates and the birth of healthy offspring (
      • Neri Q.V.
      • Lee B.
      • Rosenwaks Z.
      • Machaca K.
      • Palermo G.D.
      Understanding fertilization through intracytoplasmic sperm injection (ICSI).
      ). The AGT-revised protocol reduced the number of chemicals to the more conventional calcium ionophore, affording superior fertilization (45.9%; P<.05) and implantation rates (27.8%; P<.05), along with the birth of healthy offspring. These latter findings concur with other reports on the good health of offspring generated by this treatment (
      • Bonte D.
      • Ferrer-Buitrago M.
      • Dhaenens L.
      • Popovic M.
      • Thys V.
      • De Croo I.
      • et al.
      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.
      ,
      • Rubino P.
      • Vigano P.
      • Luddi A.
      • Piomboni P.
      The ICSI procedure from past to future: a systematic review of the more controversial aspects.
      ).
      However, this prospective cohort study is not without limitations. Clearly there may be additional causes of sperm-related OAD aside from genetic etiologies, including acrosomal dysfunction due to pollutants and drugs (e.g., Ca2+ channel blockers) (
      • Kazazoglou T.
      • Schackmann R.W.
      • Fosset M.
      • Shapiro B.M.
      Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.
      ). In addition, we cannot state that AGT or modification of the superovulation protocol by itself would provide results without the algorithm.
      The novel approach we have presented appears to be successful. Most importantly, it emphasizes using AGT, a protocol requiring the exposure of both gametes to a chemical only in cases with a confirmed sperm-related OAD. Indeed, we observed that only 1.3% of all ICSI cases treated at our center had a nearly complete absence of fertilization, demonstrating the rarity of this condition. Furthermore, as illustrated in the decision tree flowchart (Supplemental Fig. 1), proper screening of male partners using various sperm cytosolic factor assays was crucial in confirming that a small fraction of our patient cohort had a clear sperm-related OAD. Thus, it should be emphasized that AGT should only be used in couples where its potential benefit is demonstrated.

      Conclusion

      Ours is the first study to attempt to identify a gamete-specific cause of failed fertilization with ICSI even when an adequate number of oocytes and sufficient spermatozoa are present. The purpose of this investigation was to specifically identify those cases due to an oocyte- or sperm-related OAD. This was done by a PLCζ screening assay. When ooplasmic dysmaturity was the culprit, couples were successfully treated by tailoring the superovulation protocol. For the couples whose male partner had a positive screening assay, a confirmatory bioassay and a molecular genomic evaluation were also performed. We found that AGT was successful in couples where there was a clear sperm-related OAD. Indeed, its application improved fertilization, implantation, and births of healthy offspring. The algorithm presented in this contribution endeavors to identify the specific etiology for fertilization failure, in effect to avoid unnecessary interventions.

      Acknowledgments

      The authors thank the clinicians, scientists, embryologists, and nursing staff of the Ronald O. Perelman & Claudia Cohen Center for Reproductive Medicine; and Dr. Mohamad Irani for his help with the post hoc power analysis.

      Supplementary data

      Figure thumbnail figs1
      Supplemental Figure 1Decision tree flowchart. A total of 114 couples with history of ≤10% intracytoplasmic sperm injection (ICSI) fertilization rate were identified. Of these patients, 76 continued treatment at our center, while 38 elected to go elsewhere. We performed a phospholipase Cζ (PLCζ) assay for all patients before their subsequent cycles. Those couples with a negative PLCζ assay result underwent a subsequent ICSI cycle with modified superovulation protocol due to presence of oocyte-related oocyte activation deficiency (OAD). Positive PLCζ assay results were confirmed by mouse oocyte activation test (MOAT), and for some we performed a genomic assessment to identify the genes involved. These couples then underwent subsequent ICSI cycles with either the initial assisted gamete treatment (AGT) or revised AGT protocol.
      Figure thumbnail figs2
      Supplemental Figure 2Phospholipase Cζ (PLCζ) assay. A total of 52 patients tested negative for the PLCζ assay, indicating that the cause of fertilization failure of their cycles was due to ooplasmic dysmaturity. These couples underwent 105 subsequent intracytoplasmic sperm injection (ICSI) cycles with a modified stimulation protocol, yielding an improved fertilization rate of 59% (470 of 796; P<.0001). Twenty-four patients tested positive for the PLCζ assay, thus confirming a sperm-related oocyte activation deficiency (OAD). These couples underwent ICSI with either initial assisted gamete treatment (AGT) or revised AGT, revealing a fertilization rate trending upward from 37.6% to 45.9%, both more favorable than in the index cycles performed without either AGT protocol (P<.05).

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