Gabor Huszar M.D., a Senior Research Scientist, is an accomplished investigator in several areas of Reproductive Science. He has pursued research first at Harvard University and subsequently in our Yale Department of Obstetrics, Gynecology and Reproductive Sciences where he also completed the Ob/Gyn Residency.
Dr. Huszar’s interests have included the (a) Biosynthesis of muscle proteins, (b) Uterine physiology and the treatment of preterm labor. In line with this interest, Dr. Huszar has edited a book on the “Physiology and Biochemistry of the Uterus in Pregnancy and Labor.” His group has published the first paper on calcium channel blockers in the modulation of labor. Calcium channel blockers are today the first line drugs in the treatment of preterm labor. (c) Since the establishment of the IVF program at Yale, he has directed the Sperm Physiology Laboratory at the Yale Fertility Center. Dr. Huszar also participates in the treatment couples with male fertility, men who require sperm cryopreservation and couples/women who wish donor sperm.
In his translational research, Dr. Huszar has contributed a novel approach for sperm fertility assessment, and an improved IVF sperm selection method. These assays were approved by the FDA and available worldwide and at the Yale Sperm Laboratory.
Dr. Huszar is member of the Society for Gynecologic Investigation, the American Society of Reproductive Medicine, the Society of Andrology, and the European Society of Human Reproduction and Embryology. He was invited to the Editorial Boards of Biology of Reproduction, Journal of Andrology, Journal of Assisted Reproduction and Genetics, and most recently, the Journal of Women’s Health and Infectious Diseases. Dr. Huszar has also served on the Reproductive Biology Study Section of the NIH. He has published over 120 papers and was invited speaker over 80 National and International Congresses. He received the Male Reproduction and Urology Prize Paper award from the ASRM, as well as the President’s Prize from the Society for Gynecologic Investigation.
Further honors include membership in the Hungarian Academy of sciences and the Connecticut Academy of Sciences, invitations as speaker to the Nobel Committee Conference, “The Reproduction in the 21st Century meeting “Keynote Speaker of the Alpha Society. Also, Distinguished Invited Speaker of the Japanese Society of Assisted Reproduction, of the Hong Kong Society of Reproduction, of the 2012 London Alpha Society meeting, at the 2012 meeting of the British Andrology Society and of the Serono Symposia in Brazil.
Further Research Interests
Our main Research Interest (eight representative publication summaries) are listed by topics. The projects are a combination of Basic Bench Research (objective biochemical markers and sperm physiology) and Translational Research (with Clinical aspects related to male fertility, male fertility testing, and selection of the best sperm for IVF – ICSI)
Alberto Davila, Shannon Kostin, Jill Stronk and Gabor Huszar. The sperm-hyaluronic acid (HA) binding test is a sperm biomarker that reflects sperm development, and measures sperm function and fertilizing potential. Sperm-HA binding ability develops as a consequence of spermatogenetic and spermiogenetic events that are reflection of testicular function and respective endocrine regulation in men. Sperm-HA binding is also related to sperm DNA chain integrity, low sperm aneuploidy frequency, and the zona pellucida-binding ability of spermatozoa. The HA-binding score is largely independent from seminal sperm concentrations, and it is a stable semen attribute (Huszar et al., Reproductive Biomedicine Online, 2007). We are exploring the potential of sperm-HA binding test, in the early detection of decline in testicular function, inception of Andropause and related loss of function in testicular events that are necessary for normal sperm development.
Conclusions: The sperm HA-binding score reflects well testicular function with respect sperm production and proportion of sperm with zona pellucida binding ability. In the 768 men, we also detected an age related decline in high proportion (>65%) of HA binding sperm, and an increase of samples with low HA-binding score (<30%).
“Biochemical markers for the detection of human sperm with diminished development: Advances in the Andrology and Embryology laboratories due to benefits of the sperm-HA-binding”
In the past two decades various sperm biomarkers have been identified by the Huszar laboratory, and their role in human sperm fertilizing potential, implantation, and pregnancy success have been studied. In the first approach, sperm creatine kinase content and the presence of surplus cytoplasm were assessed, which reflects incomplete sperm cytoplasmic extrusion during spermiogenesis. Indeed, it has been observed that normal spermatozoa which completed cytoplasmic extrusion preferentially bind to the zona pellucida. These experiments provided three conclusions: 1) Sperm that failed to undergo cytoplasmic extrusion (thus contain excess cytoplasm} are diminished in binding to the zona pellucida, 2) Sperm-oocyte interaction during fertilization is directed and regulated for the most part by the attributes of the spermatozoa. 3) Another key development has been the recognition of the sperm plasma membrane remodeling, simultaneously with cytoplasmic extrusion, in terminal spermiogenesis. This remodeling facilitates the formation of the zona pellucida and hyaluronic acid binding site(s) of sperm. Sperm that fail to undergo the plasma membrane remodeling are unable to recognize the zona pellucida, thus potential for conventional (not ICSI) fertilization of such sperm is diminished. Incomplete cytoplasmic extrusion and plasma membrane remodeling were linked to upstream spermatogenetic and spermiogenetic defects which may adversely affect chromatin development, DNA integrity, and frequency of chromosomal aneuploidies.
The observations regarding the common origin of the zona pellucida and hyaluronic acid receptor led to two inventions: 1) For the first time in the history of Andrology, there is now a semen test (in addition to assessment of sperm concentration, motility and morphology), which predicts what percent of sperm in an ejaculate would have zona binding properties. This is a very important opportunity for physicians and patients. 2) The individual hyaluronic acid bound spermatozoa are removable with a micropipette and may be used for intracytoplasmic sperm injection. This provides the opportunity to the embryologist to have ICSI fertilization with sperm which normally would have been fertilized in the zona pellucida mediated physiological circumstances during intercourse or IVF.
Leyla Sati, M.S.1,4, Laszlo Ovari, M.D.2, David Bennett, B.S.1, Stephen D. Simon, Ph.D.,3, Ramazan Demir, Ph.D.,4, and Gabor Huszar, M.D.1 (Reproductive Biomedicine Online, 2008)
We studied attributes of arrested maturation in human spermatozoa with assessment of individual sperm with probe combinations for persistent histones and cytoplasmic retention, persistent histones and DNA degradation, persistent histones and apoptotic markers, as well as persistent histones and strict morphology.
Individual spermatozoa were assessed sequentially with combinations of cytoplasmic and nuclear biochemical markers. The sperm fields were recorded with computer assisted imaging, and the staining patterns with the two probes were scored in both images of the respective spermatozoa, as negative, intermediate and dark (mature to arrested maturity spermatozoa). There was a close correlation (>80% agreement) between the staining patterns of probe combinations in individual spermatozoa. Thus, the cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, 16% of patterns were discordant, thus testing with single maturity markers may be uncertain.
These findings are important from the viewpoints of a) arrested sperm maturation; b) potential efficacy of anti-oxidant and similar therapeutic strategies in subfertile men, as sperm with arrested maturation, apoptosis and related spermatogenetic defects are unlikely to respond to reproductive intervention; and c) relevance of adverse environmental exposures in men.
(Fertility and Sterility, 2010) Sati G1,3, Sakkas D2, Ovari L1, Fejes I1, Demir R3, and Huszar G1
Diminished sperm maturity, which affects the paternal contribution of sperm to the zygote, may be detected by various biochemical markers, including aniline blue staining, which detects the presence of persistent histones (1,2). We hypothesized that spermatozoa with retarded histone-protamine replacement might be vulnerable to apoptosis. Thus, we studied the same spermatozoa with combinations of aniline blue staining and DNA nick-translation (DNA fragmentation) and with aniline blue staining and immuno-staining for the active caspase-3 apoptotic enzyme (after capturing the aniline blue sperm images, the sperm were de-stained and the second probe was applied). We focused upon the detection of persistent histones in order to potentially identify individual sperm that are potentially of diminished paternal contribution, and thus aniline blue would facilitate the prospective evaluation of men prior to assisted reproduction therapies (3,4).
In this three-step study, first we stained sperm smears with aniline blue, and ascertained whether the staining pattern of individual sperm was light (L), intermediate (IN) or dark (D). Further, we captured the images of sperm fields and individual sperm with MetamorphTM (Universal Imaging Co. Downingtown PA). Second, we de-stained aniline blue stained sperm, and further treated the same spermatozoa with probes for DNA nick translation and active caspase-3 immuno-staining. Capturing the respective fields and sperm allowed the comparison of staining patterns. Third, we performed morphometry on spermatozoa having various aniline blue staining intensity.
We have examined the relationships between persistent histones due to delayed histone-protamine replacement (aniline blue staining, ref. 3,4) and two other markers, DNA fragmentation and apoptosis, in the same sperm that are known to affect the paternal contributions.
Study of the same sperm with aniline blue staining and DNA nick translation indicated that 84.4% of the cells were comparable in light-light, intermediate-intermediate and dark-dark staining patterns. Thus, aniline blue staining also reflects the integrity status of DNA strands in spermatozoa. Aniline blue staining was also related to the apoptotic process, as 86.3% of spermatozoa followed the light-light, intermediate-intermediate and dark-dark patterns with both aniline blue and caspase-3. Thus, these objective sperm biochemical markers facilitate the assessment of the paternal contribution of the sperm to the zygote.
From the perspective of male fertility evaluation and paternal contribution of sperm, the aniline blue intermediate sperm (approximately 15-20% in our moderately oligozoospermic population) may have major importance. This is because the immature dark sperm are unlikely to fertilize and create a zygote. However, the intermediate sperm, which do not have obviously abnormal dimensions, but carry lower levels of caspase-3 and consequential DNA fragmentation may initiate conception and zygote formation with the fragmented DNA, however the maintenance of zygote development and pregnancy are uncertain. The aniline blue intermediate sperm may represent a yet unidentified cause of unexplained male infertility.
Leyla Sati, Sevil Cayli, Elena Delpiano, Denny Sakkas, , and Gabor Huszar, (Reproductive Sciences, 2013)
In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed by others, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-relatedmanner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck,regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of spermactivation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.
N. Geerts1, J. McGrath2, J.N. Stronk3, T.K. Vanderlick1 and G. Huszar3
In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, we propose a new delivery strategy. Here we show that spermatozoa can transport vesicles of variable composition. For human sperm, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with either positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant ‘on’ and ‘off’ binding. The physiological and motility attributes of the spermatozoa are not affected by attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum Sativum Agglutinin, was not affected by vesicle binding. Moreover, the sperm hyaluronic acid binding test showed that viable, fully developed sperm will attach and remain bound to hyaluronic acid coated slides, regardless of vesicle binding. Therefore a new “hybrid” delivery system is created with human sperm, and tested with a mouse in vitro fertilization system. Large unilamellar vesicles physisorbed to mouse sperm can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles. These key findings in human sperm-vesicle hybrids were followed with proof-of-principle in vitro fertilization (IVF) experiments in the mouse-model, to determine if vesicle loaded sperm is capable of binding and penetrating the oocyte. First the presence of fluorescence, originating from the lipids of the vesicle membrane, is used to determine that transport of the vesicles into the oocyte is feasible. Second, the vesicles are filled with an (messenger) mRNA-GFP (GFP: green fluorescent protein) construct (cargo), in order to confirm that transport and delivery of the cargo within the vesicles is accomplished. This system may be applicable to genetic therapy.
Petra Prinosilova1, 2, Thinus Kruger3, Leyla Sati1, 4, Sinan Ozkavukcu1, Lynne Vigue1, Ertug Kovanci5 and Gabor Huszar1 (RBM Online, 2009)
During spermiogenesis a plasma membrane remodeling step facilitates formation of sperm zona pellucida and hyaluronic acid (HA) binding sites. We hypothesized an enrichment of Tygerberg normal spermatozoa in HA-bound sperm vs. semen sperm fractions. Semen was placed on the “A” plain glass side and HA-coated “B” side of a semen chamber. After 15 minutes, the HA-binding score (the proportion of motile sperm bound to HA) was assessed on the “B” side, and the unbound spermatozoa were removed by gentle rinsing. Following Diff-Quick staining, sperm morphology was evaluated on the both sides by three blinded investigators at Yale and Tygerberg. The proportion of Tygerberg normal sperm was higher in HA-bound vs. the semen sperm fraction (N=63 subjects). There was a 3.04x improvement (95%CL:1.9-4.7x) in 37 teratozoospermic men, comparable to the 4.2-fold enrichment in zona pellucida-bound vs. semen sperm reported earlier. The selection power of HA and zona pellucida for normal spermatozoa are comparable. There were investigator-to-investigator variations in strict morphology scores, however this reflected differences in perception of normality, as scores of the three investigators were correlated. Regarding the maturity biomarkers of sperm creatine phosphokinase activity (measure of retained cytoplasm in arrested maturity sperm) and key chaperon protein HspA2, they were proportional with HA-binding. As HA-binding reflect sperm maturity and function, the combination of Tygerberg morphology and HA-binding tests likely to improve male infertility management.
J.of Andrology, 2010. Artay Yagci1,3, William Murk2, Jill Stronk1, and Gabor Huszar1,4
During spermiogenesis, human sperm undergoes a plasma membrane remodeling step which facilitates the formation of the zona pellucida and hyaluronic acid(HA) binding sites. Studies with various biochemical sperm markers directed to the cytoplasmic and nuclear compartments indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA chain fragmentation, normal sperm shape and low frequency of chromosomal aneuploidies. In other line of experiments, zona pellucida-bound sperm studied with acridin orange fluorescence (AOF) were of high DNA integrity exhibiting green AOF. In the present work, we tested the hypothesis that HA-bound sperm, similar to zona pellucida-bound sperm, would also be enhanced in sperm with high DNA chain integrity and green AOF.
In 50 men, we have studied AOF in unselected semen sperm and in their respective HA-bound sperm fractions. The proportions of sperm in semen with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9±2.0% and 45.0±1.9%, whereas in HA-bound sperm fraction, the respective proportions were 99% and 1.0%. The data suggest that zona pellucida and HA-bound sperm show similar degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, function, and sperm selection for fertilization via intracytoplasmic sperm injection.
In earlier sperm DNA integrity studies with AO, chromatin structure of zona pellucida-bound spermatozoa was examined in sperm removed from zona pellucida of unfertilized oocytes (Liu and Baker, 2007). The authors reported > 85% frequency of sperm with green AOF reflecting high DNA integrity. In the present study, we examined the hypothesis that HA-bound sperm, similar to zona pellucida-bound sperm, will be enriched in sperm with high DNA integrity green AOF compared to their respective semen fractions. In addition to the similar attributes of HA-selected and zona pellucida-bound sperm fractions, this study is relevant from the clinical aspects. The Andrology testing with the sperm-HA binding assay does reflect the proportion of sperm in semen that would be able to bind to the zona pellucida. The almost exclusive presence of sperm with green AO fluorescence support the benefits of HA-mediated sperm selection which facilitates ICSI treatment with sperm that would have been selected by sperm-zona pellucida binding during natural or conventional IVF fertilization. (Kruger, et al., 1986;Huszar and Vigue, 1994; Gergely, et al., 1999;Huszar, et al., 2003;Celik-Ozenci, et al., 2004; Huszar, et al., 2007).
- Sperm micro RNA Sperm cryopreservation (new media, no biological components)
- Sperm Viability test Sperm Migration test (efficiency)
- Sperm select research (heat-cold exposure)
- Next day measurement of sperm motility by mitochondrial activity
Ali Rustu Ergur