Polygenic embryo screening: four clinical considerations warrant further attention.

Recent advances in developing polygenic scores have made it possible to screen embryos for common, complex conditions and traits. Polygenic embryo screening (PES) is currently offered commercially, and though there has been much recent media and academic coverage, reproductive specialists' points of view have not yet been prominent in these discussions. We convened a roundtable of multidisciplinary experts, including reproductive specialists to discuss PES and its implications. In this Opinion, we describe four clinically relevant issues associated with the use of PES that have not yet been discussed in the literature and warrant consideration.

Localizations of intracellular calcium and Ca2+-ATPase in hamster spermatogenic cells and spermatozoa.

Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37 degrees Celsius for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca(2+)-ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca(2+)-ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca(2+)-ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis.

New hope for infertility therapy: fabricating gametes from stem cells.

The field of reproductive and developmental biology has been revolutionized by recent advanced studies. These studies indicate that stem cells are capable of forming gametes in vivo and in vitro in both mouse and human. This has provided powerful tools for undertaking new types of reproductive studies, and particularly might provide new technology and novel approaches in assisted reproductive medicine.

Fertilization abnormalities following human in vitro fertilization and intracytoplasmic sperm injection.

Fertilization abnormalities are commonly encountered following in vitro fertilization. The widespread introduction of assisted fertilization methods has rapidly led to a changes in both the incidence and types of these aberrations. Such abnormalities can be identified by careful morphological evaluation at the early zygote stage, of pronuclei as well as of polar body formation, and may be confirmed by cytogenetic assessment. The recognition and understanding of fertilization abnormalities have led to the development of novel techniques aimed at preventing them, as well as recent bold attempts at correction. Removal of one pronucleus may allow some triploid embryos to revert to a normal diploid stage. These new developments should provide insight into the understanding of parthenogenesis, androgenesis, and gynogenesis in the human. Microsc. Res. Tech. 61:358-361, 2003.

Metal ions and human sperm mannose receptors.

Zinc and lead concentrations were measured in seminal plasma from fertile donors, infertile men with varicocoele and men undergoing work-ups for in vitro fertilization. Ejaculated spermatozoa from these subjects were incubated in vitro with various metal ions and/or dibromoethane and dibromochloropropane. Mannose receptor expression was correlated with metal and toxicant levels. Sperm distributions of potassium channels were compared with lead ions and calcium channels with zinc ions. Mannose receptor expression by capacitated spermatozoa increased linearly with seminal plasma zinc levels, and correlated inversely with lead levels. Cobalt had no effect on mannose receptor expression, but nickel had a concentration-dependent biphasic effect. Mannose receptor expression was not affected by dibromoethane and dibromochloropropane if the cholesterol content of the sperm membrane was high, but mannose receptor expression was decreased in low cholesterol spermatozoa by exposures below estimated permissive exposure limits. Potassium channels and lead ions co-localized over the entire head of human spermatozoa, while both calcium channels and zinc ions were confined to the equatorial segment of the head. Mannose receptor expression on the external surface of the human sperm plasma membrane is a biomarker for the effects of transition and heavy metals and organic toxicants on sperm fertility potential.

Is chemiluminescent immunoassay an appropriate substitution for radioimmunoassay in monitoring estradiol levels?

OBJECTIVE: To determine the correlation between serum estradiol measurements by chemiluminescent immunoassay (CIA) vs. radioimmunoassay (RIA) in two groups: patients treated with gonadotropins and patients treated with oral estrogen. DESIGN: Prospective study. SETTING: Assisted Reproductive Technology (ART) program based in a university-affiliated hospital in Manhasset, New York. PATIENT(S): Three hundred forty-eight patients undergoing gonadotropin stimulation and 63 patients receiving oral estrogen between July and December, 1997. INTERVENTION(S): Estradiol levels were measured concomitantly on all patients undergoing gonadotropin stimulation for IVF and all patients receiving oral estrogen for a frozen-thaw cycle. MAIN OUTCOME MEASURE(S): RIA:CIA ratio. RESULT(S): In the group undergoing gonadotropin stimulation, the median RIA:CIA ratio was 0.92, RIA = 1.26 x CIA(0.96), r = 0.98. In the group receiving oral estrogen, the median ratio was 3.93, RIA = 2.9 x CIA(1.05), r = 0.89. CONCLUSION(S): Estradiol levels determined by CIA correlate closely with RIA results for patients being treated with gonadotropins. Conversely, for patients receiving oral estrogen, CIA levels are one-third or less of the RIA level.

Numerical dose-compensated in vitro fertilization inseminations yield high fertilization and pregnancy rates.

OBJECTIVE: To evaluate in cases with morphologically abnormal sperm whether fertilization and pregnancy rates are increased by normalizing the number of sperm inseminated and whether biomarkers can identify cases of reduced or failed fertilization. DESIGN: Prospective studies of sperm morphology and function. SETTING: University hospital assisted human reproduction program. PATIENT(S): Partners of 308 women undergoing IVF. INTERVENTION(S): Motile sperm populations were assessed for sperm head morphology, for surface receptors for mannose and progesterone binding, and the ability to undergo a free mannose-induced acrosome reaction. Zinc in seminal plasma was determined by atomic absorption spectroscopy. MAIN OUTCOME MEASURE(S): Sperm morphology was associated with fertilization and clinical pregnancy rates. Biomarker analyses were correlated with fertilization rates using Kruskal-Wallis tests, chi2 tests, and Spearman rank order correlations. RESULT(S): Fertilization and pregnancy rates after numerical dose compensation inseminations were indistinguishable between men with differing percentages of normal sperm. Biomarker deficits were identified irrespective of sperm head morphology in 96% of cases of reduced or failed fertilization. CONCLUSION(S): Fertilization and pregnancy rates in cases of abnormal morphology are optimized by inseminating at least 25,000 sperm/mL with normal acrosomes. Reduced or failed fertilization can be predicted by testing for molecular deficits in mannose receptor expression and mannose-stimulated acrosome loss.

Monozygotic twinning associated with mechanical assisted hatching.

OBJECTIVE: To determine the effect of mechanical assisted hatching on the pregnancy rate (PR). DESIGN: A retrospective comparative analysis of hatched versus nonhatched consecutive assisted reproductive technology (ART) cycles. SETTING: A hospital-based ART program. PATIENT(S): Patients undergoing ART treatment with assisted hatching (1994-1996) were compared with patients who did not have assisted hatching (1990-1993). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate, multiple PR, and rate of monozygotic twinning. RESULT(S): With hatching, the clinical PR per ET increased from 25.2% to 37.1% and the multiple PR per ET increased from 6.8% to 13.1%. In the nonhatched series, there were no monozygotic twins compared with eight cases in the hatched series (1.2% per ET). CONCLUSION(S): Mechanical assisted hatching increases the PR but concomitantly elevates the rate of multiple gestation and multiple gestation of high order. There is a particularly high risk of monozygotic twinning with mechanical hatching.

Mannose ligand receptor assay as a test to predict fertilization in vitro: a prospective study.

OBJECTIVE: To assess whether mannose receptor assays can predict fertilization outcome in vitro. DESIGN: A prospective, double-blind study of the mannose receptor properties of spermatozoa. SETTING: Assisted human reproduction program at a university hospital. PATIENT(S): Partners of 140 consecutive women undergoing their first in vitro fertilization cycle. INTERVENTION(S): Motile sperm populations were tested for surface receptors for mannose by measuring their ability to bind fluorescein-labeled mannosylated albumin and to undergo a free mannose-induced acrosome reaction as judged by Pisum, sativum agglutinin binding. MAIN OUTCOME MEASURE(S): Mannose receptor assay results were correlated with fertilization outcomes using several statistical tests, including the chi2 test, chi2 for proportions, t-tests, analysis of variance with Student-Newman-Keuls tests and correlational and receiver operating characteristic (ROC) curve analysis. RESULT(S): The fractional increment increase on incubation in the percent of sperm binding mannose ligand over an intact acrosome correlated with fertilization rates in vitro. Threshold values of mannose ligand binding and of mannose-induced acrosome reactions predictive of fertilization rates were identified by ROC curve analysis. Men were thus classified into one of four groups with differing fertilization rates in vitro. CONCLUSION(S): The increment increase in sperm surface mannose ligand binding by acrosome-intact sperm correctly predicts high and low fertilization rates in vitro and identifies cases where conventional insemination can result in failed fertilization.

Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome.

In a prospective, blind study, we have examined the relationship among the expression of human sperm surface progesterone receptors, the ability to undergo a mannose-stimulated acrosome reaction and the rate of fertilization in vitro. Individual aliquots of motile spermatozoa were surface-labelled with progesterone and/or mannose-fluoresceinated ligands. Spontaneous acrosome loss and the increase in acrosome reactions following exposure of spermatozoa to mannose ligands were assessed using rhodaminated Pisum sativum agglutinin. Progesterone fluoresceinated ligand binding was observed to occur in two patterns: (i) a uniform distribution of labelling over the acrosome cap (pattern II), and (ii) labelling limited to the equatorial and postacrosomal regions of the human sperm head (pattern III). A conversion of pattern II to pattern III binding was observed and was associated with the acrosome reaction. Pattern III binding was highly correlated with both fertilization potential and the ability to undergo a mannose-stimulated acrosome reaction (P < 0.001). In contrast, normal sperm mannose receptor expression was seen in five men whose abnormal progesterone receptor expression/function and inability to acrosome react after mannose treatment were correlated with their reduced fertility in vitro. In conclusion, surface progesterone receptor aggregation enhances the mannose ligand-stimulated acrosome reaction. Such detection of defective sperm surface progesterone receptor expression/function may be useful in the evaluation and management of male infertility.

Induction of the human sperm acrosome reaction with mannose-containing neoglycoprotein ligands.

In the interest of classifying cases of male factor infertility, we have paid particular attention to the sugar ligand binding properties of the human sperm surface and the functional capacity of the acrosome for exocytosis--key parameters for assessing sperm fertilizing ability. Zona recognition and binding involve the interactions of sperm surface mannose receptors (lectins) with mannose ligands on the zona pellucida. Sperm surface mannose lectins can be visualized by their ability to bind a synthetic model zona ligand, fluorescein isothiocyanate (FITC)-conjugated mannosylated bovine serum albumin (BSA) (Man-FITC-BSA). We now report that Man-FITC-BSA biologically also mimics the effects of solubilized authentic human zonae, in that binding of Man-FITC-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors. In our assay, the addition of mM amounts of mannose monosaccharide to Man-FITC-BSA increases the number of polyvalent mannose ligands bound by individual spermatozoa and increases the rate of the acrosome reactions induced by Man-FITC-BSA, thereby increasing specimen processing efficiency. We conclude that exposure of human spermatozoa to polyvalent mannose ligands + D-mannose monosaccharide offers a new, convenient and readily available system to study sperm capacity for induced acrosome loss.

Use of mannose ligands in IVF screens to mimic zona pellucida-induced acrosome reactions and predict fertilization success.

A predictive test for determining whether motile populations of human spermatozoa will fertilize eggs in vitro has been an elusive goal of clinical research. We have developed an assay for the ability of motile human spermatozoa to bind fluorescein isothiocyanate-labelled mannosylated bovine serum albumin (Man-FITC-BSA) as a test for the presence of sperm surface receptors (lectins) for mannose ligands. Mannosylated ligands are present on the human zona pellucida and are involved in the species-specific binding of human spermatozoa to the zona pellucida. We now demonstrate in prospective blinded analysis that the fractional increase in acrosome loss following a mannose ligand challenge is highly correlated with the rate of fertilization in vitro. Using an incremental increase of acrosome exocytosis of >0.1 as a threshold to predict which specimens will yield normal fertilization, the assay has a sensitivity of 97.8%, a specificity of 83.3%, a positive predictive value of 95.7% and a negative predictive value of 90.7%. These data indicate that testing for a mannose-induced acrosome reaction may be useful in assessment of sperm function prior to in-vitro fertilization in order to assign males to conventional insemination or intracytoplasmic sperm injection protocols.

Acrobeads test as a predictor of fertilization in vitro.

PROBLEM: To determine whether the results of the Acrobeads test, which measures the expression of the complement regulator molecule CD46 on the inner acrosomal membrane following the acrosome reaction, accurately identifies semen specimens that will exhibit reduced or failed fertilization following conventional IVF insemination. METHOD: The Acrobeads test was performed on semen specimens from 97 consecutive patients preparing to undergo an IVF cycle utilizing a standardized insemination protocol. Motile sperm populations were examined at 6 h and 24 h post-isolation for sperm-bead agglutination. Results of the Acrobeads test were compared to that of TRITC-PSA staining in matched specimens to directly measure the spontaneous loss of acrosome content. The percentages of TRITC-PSA-negative sperm were determined in freshly isolated motile populations and in duplicate aliquots incubated 18 to 20 h under sperm capacitating conditions. The relationship between the results of both analyses estimating spontaneous acrosome reactions and the rate of fertilization of metaphase II oocytes was examined. RESULTS: The Acrobeads score did not correlate significantly with the rate of fertilization by insemination at 6 h or at 24 h. The negative predictive value of this test was 21.4%. There was no correlation between the Acrobeads score and the percentage of sperm undergoing a spontaneous acrosome reaction as detected by TRITC-PSA labeling. In contrast, the increment increase in the percentage of spontaneous acrosome reactions as quantified by TRITC-PSA staining was correlated with the fertilization rate. CONCLUSIONS: Contrary to previous reports, our prospective, double-blinded study failed to demonstrate that the Acrobeads test can accurately predict fertilization outcome in IVF. Therefore, the routine use of this test to screen patients prior to an IVF cycle in order to select appropriate treatment (i.e., ICSI) cannot be recommended.

A potential role for cadmium in the etiology of varicocele-associated infertility.

OBJECTIVE: To determine whether mannose ligand receptor and acrosome reaction deficits in sperm from men with varicocele are related to the transition metal content of their semen. DESIGN: Cadmium and zinc in semen and blood plasma were assayed for fertile males, men without varicocele who required intracytoplasmic sperm injection to achieve fertilization, and men evaluated for potential varicocele-associated infertility. The relationship between actin cytoskeletal distributions and acrosome status was determined for fertile donor sperm in the presence and absence of exogenous cadmium. SETTING: University hospital-based molecular biology research laboratory. PATIENT(S): Patients from two university hospital-based IVF-assisted reproductive technology programs and two male urology private practices. INTERVENTION(S): Fertile donor sperm were exposed to exogenous cadmium during capacitating incubations followed by culture at temperatures up to 41 degrees C. MAIN OUTCOME MEASURE(S): Metal ion levels in semen and blood plasma were determined by graphite furnace atomic absorption spectroscopy. Motile sperm were examined for mannose ligand binding and the ability to undergo spontaneous and induced acrosome reactions. Unfixed, Triton-permeabilized sperm were probed with antiactin and antimyosin antibodies. RESULT(S): Cadmium was elevated and zinc was decreased in the seminal plasma of men with varicocele. The content of these metals in semen and blood was not correlated. Cadmium exposure in vitro reduced mannose receptor expression, acrosome exocytosis, and cytoskeletal formation by fertile donor sperm. CONCLUSION(S): Defects in transition metal regulation or excessive cadmium exposure are involved in varicocele-associated infertility.

Classification of male factor infertility relevant to in-vitro fertilization insemination strategies using mannose ligands, acrosome status and anti-cytoskeletal antibodies.

Polyvalent mannose ligands in the presence of free mannose act as zona pellucida agonists which rapidly induce acrosome exocytosis in competent motile human sperm from fertile donors following in-vitro capacitation. Quantification of the binding patterns of fluorescein isothiocyanate-labelled mannosylated albumins and of specific antisera which recognize mannose receptors and other related integral sperm membrane proteins as well as the incidence of induced acrosome exocytosis after capacitation has allowed us to identify three categories of male infertility. Category 1 males have normozoospermic semen parameters, their spermatozoa have elevated sperm cholesterol values and fail to fertilize oocytes in vitro after standard short-term incubations. These spermatozoa do not bind mannose ligands and do not show spontaneous or induced acrosome reactions, but treatments to remove cholesterol from the spermatozoa (e.g. prolonged incubation in the presence of sterol acceptors) confer the ability to fertilize. Cholesterol loading and unloading experiments have demonstrated the reversible character of sperm membrane properties in category 1 male infertility. Category 2 males have normal-appearing spermatozoa in semen which express mannose ligand receptors on incubation, but fail to undergo acrosome reactions in response to mannose treatment. Interestingly, all category 2 males identified in this study have clinical varicocele. Category 3 males have semen which may be normozoospermic or teratozoospermic with, in some cases, high percentages of tapering spermatozoa in the absence of clinical varicocele. Spermatozoa from category 3 men are deficient in a superfamily of integral membrane proteins whose cytoplasmic tails have myosin motors as identified by amino acid sequence analysis and anti-myosin antibody reactivity. Their spermatozoa do not express mannose ligand receptors or undergo induced acrosome reactions. Fertilization with category 2 and 3 semen is only achieved by micromanipulation procedures. These findings illustrate the practical application of basic research for infertility classification.

Co-expression of mannose-ligand and non-nuclear progesterone receptors on motile human sperm identifies an acrosome-reaction inducible subpopulation.

PROBLEM: To determine whether surface expression of receptors for progesterone and mannose can be used to identify spermatozoa likely to undergo an acrosome reaction after zona binding and to compare the reactivity of these receptors with naturally occurring sperm head-directed anti-sperm antibodies (ASAs). METHOD: Progesterone binding sites on the surface of fresh and capacitated motile human sperm in relation to acrosome status were visualized using a cell-impermeant progesterone. Free progesterone and/or mannose ligands were compared for percent sperm binding and ability to induce an acrosome reaction. Western blots of sperm proteins localized to the plasma membrane and surface proteins precipitated following passive transfer of serum ASAs were probed with progesterone-horseradish peroxidase. The effects of the same ASAs on ligand binding and on the induced acrosome reaction were examined. RESULTS: The two receptors are located in close proximity on a subset of capacitated motile sperm and are coordinately cleared from the plasma membrane overlying the acrosomal cap prior to exocytosis. The surface appearance of functional binding sites for each ligand, however, is regulated by different mechanisms and the progesterone receptor alone is specifically precipitated by ASAs. Passive transfer of ASAs to capacitated sperm selectively inhibits the progesterone-stimulated acrosome reaction but not the ionomycin-induced acrosome reaction or the ability of sperm to bind mannose ligands. CONCLUSIONS: Sperm from fertile donors incubated under capacitating conditions in vitro can be subdivided into acrosome reaction inducible and noninducible subpopulations on the basis of the co-expression or total absence of these receptors. The combined data indicate that reaction of sperm surface progesterone receptors with ASAs contributes to the acrosome reaction insufficiency observed in anti-sperm immune infertility.

Pregnancy following discontinuation of a calcium channel blocker in the male partner.

The fertility potential of human sperm populations can be assessed by the presence of head-directed mannose ligand receptors (mannose-specific lectin) and the occurrence of spontaneous acrosome reactions after incubation under capacitating conditions in vitro. We have reported previously on the interaction between anti-hypertensive medications and their effects on these parameters of male fertility potential. In this report we document the effects of cessation of calcium ion channel blocker medication on male fertility. Motile spermatozoa from a 30 year old infertile patient on a calcium ion channel blocker as anti-hypertensive treatment had subnormal expression of mannose-specific lectin and did not exhibit spontaneous acrosome reactions. Three months following discontinuation of the medications, complete recovery of both the expression of head-directed mannose ligand receptors and the acrosome reaction was documented, though sperm motility and morphology remained unchanged. The couple had 2 years of infertility and previously failed to conceive through seven cycles of Pergonal/intra-uterine insemination. Conception occurred on the second Pergonal/intra-uterine insemination cycle after the husband discontinued calcium ion channel blocker medication. Calcium ion channel blockers may adversely affect sperm fertilizing potential. Discontinuation of such medications enhances the changes for conception.

The effect of calcium ion channel blockers on sperm fertilization potential.

OBJECTIVE: To evaluate the effects of calcium ion (Ca2+) channel blockers on male fertility potential. DESIGN: A case comparison of the surface expression of mannose-ligand receptors on motile spermatozoa from 10 known fertile males and from 10 normospermic men taking Ca2+ channel blockers who were seeking infertility treatment. Examination of the effects of in vitro exposure of sperm from fertile donors (n = 14) to antihypertensive medications. SETTING: Patients from a successful university hospital-based IVF-assisted reproductive technology program and from a male urology private practice. INTERVENTIONS: Prescription of alternate hypotensive medications for four male patients; cholesterol loading and unloading in vitro of fertile donor sperm. MAIN OUTCOME MEASURES: Motile sperm were tested for their ability to bind fluorescein isothiocyanate-labeled, mannosylated bovine serum albumin as an index of the surface expression of mannose-ligand receptors associated with fertility potential. Acrosome status was simultaneously evaluated by fluorescence microscopy with rhodamine-labeled Pisum sativum lectin. Sperm were assayed before and after an 18-hour or 3-day incubation under capacitating conditions in vitro. RESULTS: Motile spermatozoa of normospermic men taking calcium antagonists for hypertension control do not express head-directed mannose-ligand receptors at high frequency, nor do they undergo spontaneous acrosome loss. Unexpectedly, mannose-ligand receptor translocation from the subplasmalemmal space over the acrosome to the sperm surface and aggregation over the equatorial-postacrosomal regions occurred in acrosome-intact sperm. This differs from fertile controls in whom receptor translocation to the equatorial-postacrosomal segment is coupled with the acrosome reaction (AR). Discontinuation of calcium antagonists results in complete recovery of parameters associated with sperm fertilizing potential: time-dependent increases in the percentages of spermatozoa exhibiting surface mannose-ligand binding and spontaneous ARs in vitro. The effects of in vivo administration of calcium antagonists is mimicked in control fertile donor sperm by inclusion of a Ca2+ channel blocker in the media employed during capacitating incubations. CONCLUSIONS: Therapeutic administrations of calcium antagonists for hypertension control cause reversible male infertility associated with an IVF failure. A mechanism of inhibition of sperm fertilizing potential through insertion of lipophilic calcium ion antagonists into the lipid bilayer of the sperm plasma membrane is consistent with our in vitro studies.