Effects of three-area laser-assisted zona thinning in 8-cell human embryos on pregnancy outcomes in vitro fertilization / 대한생식의학회지

OBJECTIVE: This study conducted a preliminary examination of the effects of three-area laser-assisted zona thinning (LAZT) during the cleavage stage of embryo development on the hatching process in human in vitro fertilization-embryo transfer (IVF-ET) with subjects of advanced female age or frozen-thawed (FT) embryos. METHODS: Eight-cell stage embryos were treated with LAZT in three areas of the zona pellucida at 120° intervals. The control group was embryos without LAZT. Of the 72 consecutive fresh cycles and the 28 FT embryo transfer cycles, the patients in 55 fresh cycles and 17 FT cycles declined LAZT, and those cycles were defined as the control group. RESULTS: In the fresh cycles, the pregnancy rates were similar in the LAZT and control groups. However, in the FT cycles, the pregnancy rate was significantly higher in the LAZT group than in the control group (45.5% in the LAZT group vs. 23.5% in the control group, p < 0.05). CONCLUSION: These results show that multi-area LAZT resulted in significantly improved pregnancy outcomes in human 8-cell embryos compared to controls.

Thinning and drilling laser-assisted hatching in thawed embryo transfer: A randomized controlled trial / 대한생식의학회지

OBJECTIVE: In frozen and thawed embryos, the zona pellucida (ZP) can be damaged due to hardening. Laser-assisted hatching (LAH) of embryos can increase the pregnancy rate. This study compared thinning and drilling of the ZP before frozen embryo transfer (FET). METHODS: Patients were randomly allocated into two groups for LAH using thinning or drilling on day 2 after thawing. Twenty-five percent of the ZP circumference and 50% of the ZP thickness was removed in the thinning group, and a hole 40 µm in diameter was made in the drilling group. RESULTS: A total of 171 in vitro fertilization/intracytoplasmic sperm injection FET cycles, including 85 cycles with drilling LAH and 86 cycles with thinning LAH, were carried out. The thinning group had a similar β-human chorionic gonadotropin-positive rate (38.4% vs. 29.4%), implantation rate (16.5% vs. 14.4%), clinical pregnancy rate (36.0% vs. 25.9%), miscarriage rate (5.8% vs. 2.4%), ongoing pregnancy rate (30.2% vs. 23.5%), and multiple pregnancy rate (7.0% vs. 10.6%) to the drilling LAH group. There were no significant differences in pregnancy outcomes between subgroups defined based on age (older or younger than 35 years) or ZP thickness (greater or less than 17 µm) according to the LAH method. CONCLUSION: The present study demonstrated that partial ZP thinning or drilling resulted in similar outcomes in implantation and pregnancy rates using thawed embryos, irrespective of women's age or ZP thickness.

Clinical assessment of the male fertility

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.

Hydrogen-rich Water Exerting a Protective Effect on Ovarian Reserve Function in a Mouse Model of Immune Premature Ovarian Failure Induced by Zona Pellucida 3 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Premature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, we explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model.</p><p><b>METHODS</b>To set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software.</p><p><b>RESULTS</b>Immune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P < 0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax/Bcl-2 ratio decreased in the model-hydrogen group.</p><p><b>CONCLUSIONS</b>Hydrogen-rich water may improve serum AMH levels and reduce ovarian GC apoptosis in a mouse immune POF model induced by ZP3.</p>

The effect of the site of laser zona opening on the complete hatching of mouse blastocysts and their cell numbers / 대한생식의학회지

OBJECTIVE: We studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts. METHODS: Mouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125). RESULTS: The rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group (69±19.3, 74±15.7, and 71±16.8, respectively; p=0.680). CONCLUSION: These findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers.

Efecto de la vitrificación de ovocitos humanos sobre la capacidad de unión y el estado acrosomal de espermatozoides humanos / Effect of the vitrification of human oocytes on the binding capacity and acrosome status of human sperm

Conocer los aspectos moleculares que acontecen en el proceso de unión de los espermatozoides humanos a la zona pelúcida (ZP) humana es uno de los grandes retos de la biología de la Reproducción. Por otra parte conocer si el proceso de fecundación puede verse afectado por la criopreservación de los gametos femeninos sigue siendo otra cuestión debatida en la literatura. En base a esto, el objetivo principal de este trabajo fue conocer si la vitrificación ovocitaria puede alterar la interacción de los espermatozoides con el glicocáliz de la ZP y demostrar si la ZP de estos ovocitos pierde la capacidad de inducir la reacción acrosómica en los espermatozoides. Según nuestros resultados el método de vitrificación ovocitaria cerrado (S3) no altera la capacidad de unión de los espermatozoides a la zona pelúcida, ni la capacidad de ésta para inducir la reacción acrosómica.

To know the molecular aspects that occur in the process of human sperm binding to the human zona pellucida (ZP) is one of the great challenges of reproduction biology. Moreover knowing if the fertilization process may be affected by cryopreservation of female gametes is still another issue discussed in the literature. Based on this, the main objective of this study was to determine whether the oocyte vitrification may alter the interaction of sperm with the glycocalyx of ZP and show whether these oocytes lost the ability to induce the acrosome reaction in sperm. According to our results the oocyte closed vitrification method (S3) does not alter the ability of the sperm binding to the zona pellucida, and their ability to induce the acrosome reaction.

Effect of laser-assisted multi-point zona thinning on development and hatching of cleavage embryos in mice / 대한생식의학회지

OBJECTIVE: This study aimed to examine the effect of laser-assisted zona thinning (LAZT) at one or four-points on the blastocyst formation and hatching process in mice with respect to female age. METHODS: Eight-cell or morula embryos collected from superovulated C57BL female mice with different ages (6-11 and 28-31 weeks) were treated with LAZT at one-point (LAZT1) or four-points (LAZT4). The zona pellucida was thinned to more than 70% of its initial thickness by making two holes of 15-20 microm. RESULTS: In the young mice, LAZT resulted in a significant increase in early hatching and hatching rates compared to the control group (p<0.05). However, in the old mice, LAZT significantly increased blastocyst formation as well as early hatching and hatching compared to the controls (p<0.05). These effects were more remarkable in LAZT4 than in LAZT1 and in aged mice than in young ones. CONCLUSION: These results show that multi-point LAZT leads to a significant improvement of blastocyst formation and hatching in mice compared to controls.

Fertilization and embryo quality of mature oocytes with specific morphological abnormalities / 대한생식의학회지

OBJECTIVE: To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI). METHODS: The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB). RESULTS: The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%). CONCLUSION: The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.

Effects of M II stage oocytes zona pellucida birefringence on pregnancy outcome

OBJECTIVE@#To explore the effects of different M II stage oocytes zona pellucida birefringence on pregnancy outcome.@*METHODS@#A total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively, and randomly divided into the high zona birefringence (HZB)/HZB group, HZB/low zona birefringence (LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence. Intracytoplasmic sperm injection outcome was analyzed and compared.@*RESULTS@#The proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups (HZB/HZB group>HZB/LZB group>LZB/LZB group) (P0.05). Logistic regression analysis showed that factors affect M II stage oocytes zona pellucida birefringence were age, basal FSH level and the LH level on the day of HCG injection. Age and FSH levels were negatively correlated with the single oocyte zona pellucida birefringence; While the LH level on the day of hCG injection was positively correlated with the single oocyte zona pellucida birefringence.@*CONCLUSIONS@#The primary influence factors on M II stage oocytes zona pellucida are age, basal FSH level and the LH level on the day of hCG injection. The birefringence value of zona pellucida can affect the pregnancy outcome.

Outcomes of preimplantation genetic diagnosis using either zona drilling with acidified Tyrode's solution or partial zona dissection / 대한생식의학회지

OBJECTIVE: To review the outcomes of preimplantation genetic diagnosis (PGD) using zona drilling with acid Tyrode's solution (chemical zona pellucida drilling, chemical ZD) and those of partial zona dissection (PZD). METHODS: Clinical outcomes of seventy-one couples undergoing 85 PGD cycles from January 2005 to December 2010 were included. Blastocyst formation and the hatching rate, clinical pregnancy rate, ongoing pregnancy rate, implantation rate, and fetal gender ratio of the PZD and chemical ZD groups were compared. RESULTS: Application of PZD resulted in a significantly higher rate of clinical pregnancy (40.7% vs. 15.4%, p=0.022), ongoing pregnancy (35.6% vs. 11.5%, p=0.023), and implantation (18.1% vs. 5.7%, p=0.007) compared with chemical ZD. Among non-transferred embryos, the rate of blastocyst formation on day 5 (49.1% vs. 39.5%, p=0.016) and hatching on day 6 (47.2% vs. 26.5%, p<0.001) were also significantly higher in the PZD group. CONCLUSION: The mechanical zona dissection method showed better outcomes than chemical ZD in terms of the blastocyst development and pregnancy rate. In this study, the fact that chemical ZD was conducted in different period from mechanical method should be considered in interpreting the result.

Cysteine-Rich Secretory Proteins (CRISP) and their role in mammalian fertilization

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.

[ ultrastructural study of oocyte and zona pellucida in ovarian follicles of untreated and and metformin-treated diabetic rats subsequent to induction of experimental diabetes]

Diabetes is a metabolic disorder affecting the whole body systems including the female reproductive organs. Moreover, diabetes is an important cause of infertility. Metformin is commonly used to control hyperglycemia in patients with diabetes. This study was done to evaluate the ultrastructural changes of ovarian follicles in diabetic rats and their response to metformin. Thirty-six adult Sprague-Dawley female rats [170-210 g] were studied in three groups [Control, diabetic and metformin-treated rats]. In the second and third groups, diabetes was induced by injection of streptozotocin [45 mg/kg]. The rats in the third group were later treated by metformin monohydrochloride [100 mg/kg]. At the end of the experiment, rats were sacrificed and their right ovaries were observed under transmission electron microscope. Quantitative data were analyzed by student t-test in SAS software. In comparison with the control group, significant decreases in zona pellucida thickness and the mean number of microvilli were observed [respectively, P<0.01 and P<0.001] in diabetic rats. Significant decreases in zona pellucida thickness were also observed in metformin-treated rats [P<0.05] but changes in the number of microvilli were non-significant. The number of organelles in oocyte cytoplasm was higher and they were natural or natural-looking in metformin-treated rats versus the diabetic ones. Reduction in the number of mitochondria and their ballooning cristae were of the most noticeable changes in diabetic rats. Diabetes decreases the number of microvilli and oocyte organelles and diminishes zona pellucida thickness leading to structural changes in the organelles but metformin could improve the aforesaid conditions

effects of laser assisted hatching on pregnancy rates

For infertile women aged over 35 years, failure of the ZP [zona pellucida] to rupture is believed to be associated with a decreased implantation rate in vitro fertilization [IVF] or intra cytoplasmic sperm injection [ICSI]. In this research, laser assisted hatching [LAH] was offered to patients with advanced maternal age to evaluate a possible benefit. Nine hundred thirty two cycles of IVF/ICSI in females were analyzed. Women included in this study were allocated in 4 groups. In group I and II, embryos were cultured and transferred with and without LAH in women aged /= 35. Laser manipulations were performed using a suturn-Tm3 system using 2-3 pulses of 0.8 millisecond with 400 voltage duration. The size of the hole made in the zona was measured to be 5-10 micro m, depending on the zona thickness of each individual embryo. The performance of LAH significantly increased clinical pregnancy rates in all patients. In group I and II, the chemical [50.99% and 31.61% respectively], clinical [50% and 30.69% respectively] and multiple pregnancies [22.27% and 5.94% respectively] significantly differ between these groups. In the patients with advanced female age >/= 35 the performance of LAH significantly increased chemical [30.12%] and clinical pregnancy [27.71%] rates compared to whom without LAH [18.96% and 16.37% respectively]. Our data demonstrate in the patients who were less than 35 years old, multiple pregnancy rates were significantly increased compared to other groups who aged over 35 years old. In addition benefit of LAH in improving pregnancy rates after IVF or ICSI in women of advanced age [>/= 35] was shown

Sperm-oocyte interaction and in vitro fertilization clinical outcomes in patients with unexplained infertility / 中南大学学报(医学版)

OBJECTIVE@#To determine the sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction in patients with unexplained infertility, and to discuss the relationship between ZP-induced acrosome reaction and fertilization rate.@*METHODS@#We compared the fertilization rate and good embryo rate in patients with unexplained infertility after fertilization in 2 ways. Based on the causes of infertility, patients were divided into an unexplained infertility group (Group A) and a pure female tubal factor group (Group B). Oocytes which were obtained by super ovulation from 25 patients with unexplained infertility were randomly divided into 2 groups with conventional in vitro fertilization (IVF) (Group A1) and intracytoplasmic sperm injection (ICSI) fertilization (Group A2). The pure female tubal factor group (Group B) had conventional IVF. We conducted sperm-ZP binding and ZP-induced acrosome reaction experiments with 2 groups of men's sperms separately. We compared the number of sperm-egg binding and ZP-induced acrosome reaction rate and discussed the relationship between the ZP-induced acrosome reaction and fertilization rate, and also the fertilization rate, good embryo rate and pregnancy rate in patients with unexplained infertility after fertilization in 2 ways.@*RESULTS@#The average number of sperm-egg binding (78.29 ± 16.31) and the ZP-induced acrosome reaction rate (55.87 ± 27.69) % in Group A were lower than those of Group B [94.63 ± 6.72, (82.53 ± 17.99)%]. The difference between the average number of sperm-egg binding and the ZP-induced acrosome reaction was significant (P <0.01). The fertilization rate of Group A1 was significantly lower than that of Group B and Group A2 (P <0.01). But there was no significant difference in the good embryo rate among the 3 groups. There was no significant difference between Group A2 and B in fertilization rate and good embryo rate (P <0.05). There was no significant difference in pregnancy rate between Group A and B (P <0.05). Fertilization rate and the rate of acrosome reaction had marked positive correlation with statistical significance (r =0.932, P <0.01).@*CONCLUSION@#ZP binding and ZP-induced acrosome reaction are very important experiments in sperm function test for patients with unexplained infertility. It can not only effectively avoid no embryo transferring due to complete failure of fertilization but also get a desirable outcome of pregnancy using half-ICSI fertilization in patients with unexplained infertility.

Efficiency of laser-assisted intracytoplasmic sperm injection in a human assisted reproductive techniques program / 대한생식의학회지

OBJECTIVE: Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics. METHODS: Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LA-ICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics. RESULTS: Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients. CONCLUSION: LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.

Assisted hatching em reprodução assistida: uma meta-análise de ensaios clínicos controlados / Assisted hatching in assisted reproduction: a meta-analysis of clinical controlled trials

O objetivo desta meta-análise foi avaliar o efeito da assisted hatching (AH) sobre os resultados dos ciclos de reprodução assistida: gravidez clínica, nascimento vivo, gestação múltipla, aborto e implantação embrionária, sendo avaliados os artigos publicados em periódicos indexados ao PubMed por dois autores independentes. Foram levantados 51 ensaios clínicos controlados que avaliaram o efeito da AH, sendo 40 excluídos, resultando em 11 artigos completamente avaliados. Não houve diferença significativa na taxa de gestação clínica (44,41 versus 41,30%; p=0,19, AH versus controle, respectivamente) e na taxa de nascimento vivo (36,33 versus 34,79%, p=0,63), porém, foi identificada uma tendência de aumento na taxa de gestação múltipla (18,44 versus 15,02%, p=0,05). Também não foi identificada diferença significativa na taxa de aborto (6,66 versus 6,21%, p=0,83), mas observou-se um aumento significativo na taxa de implantação embrionária (24,32 versus 21,23%, p=0,02). A partir desses resultados, pode-se concluir que, até o momento, não existe evidência suficiente para suportar o uso da AH de rotina para ciclos de reprodução assistida com transferência de embriões frescos, uma vez que não houve aumento na taxa de gravidez clínica e/ou na taxa de nascimento vivo.

The aim of this meta-analysis was to evaluate the effect of assisted hatching on the outcome of assisted reproduction cycles: clinical pregnancy, live birth, multiple pregnancy, abortion and embryonic implantation, by assessing articles published in journals indexed in PubMed by two independent authors. Fifty-one controlled trials that evaluated the effect of assisted hatching were analyzed, and 40 of them were excluded, resulting in 11 articles fully assessed. There was no significant difference in clinical pregnancy rate (44.41 versus 41.30%, p=0.19, assisted hatching versus control, respectively), and in the live birth rate (36.33 versus 34.79%, p=0.63), but we identified a trend toward increased rate of multiple pregnancies (18.44 versus 15.02%, p=0.05). We also did not identify any significant difference in the rate of abortion (6.66 versus 6.21%, p=0.83), but a significant increase in the rate of embryo implantation was observed (24.32 versus 21.23%, p=0.02). From these results, we have concluded that, until now, there is not sufficient evidence to support the use of assisted hatching for routine assisted reproduction cycles with fresh embryo transfer, since there has not been an increase in clinical pregnancy rate and/or the rate of live birth.

Binding characteristics of sperm with recombinant human zona pellucida glycoprotein-3 coated beads.

Background & objectives: An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP), an extracellular glycoproteinaceous matrix surrounding egg, is one of the plausible causes of idiopathic infertility. It will be clinically useful to distinguish this condition from other causes of infertility. An assay system, investigating binding of human sperm with ZP glycoprotein may prove useful in this regard. We attempted to develop a simple assay system to analyse the binding of capacitated human spermatozoa to human zona pellucida glycoprotein-3 (ZP3) using baculovirus-expressed recombinant human ZP3 coated beads. Methods: Recombinant baculovirus-expressed ZP3 was purified, labelled with biotin and coated on streptavidin sepharose beads. An in vitro assay system was optimized to study binding of capacitated human sperm to ZP3 coated beads. Results: A higher percentage of baculovirus-expressed recombinant human ZP3 coated beads showed significant (P<0.05) binding of capacitated human sperm as compared to beads coated with fetuin. An inhibition in the binding of sperm to ZP3 coated beads was observed in presence of cold recombinant human ZP3. Further, prior incubation of ZP3 coated beads with monoclonal antibodies (MAbs) against ZP3 but not against ZP2 resulted in the decrease in number of sperm bound to bead. Interpretation & conclusion: An in vitro assay system to study the binding of human sperm to ZP3- primary sperm receptor was established, which may be useful to determine the functional competence of spermatozoa.

Incidence of anti-zona pellucida and anti-sperm antibodies among infertile Jordanian women and its relation to mycoplasmas

Anti-zona-pellucida autoantibodies [AZP-Ab] and anti-sperm isoantibodies [ASA] were assessed in the cervical secretions from 73 infertile Jordanian women and 41 fertile control women using latex agglutination. Significantly more women with infertility had AZP-Ab and ASA [16.4% and 8.2% respectively] compared with fertile women [9.4% and 0%], with no relation to the etiology of infertility. Using polymerase chain reaction Mycoplasma hominis and Ureaplasma urealyticum were detected in cervical secretions of 19.2% and 13.7% of infertile women, and the presence of mycoplasma was significantly correlated with the presence of AZP-Ab and ASA

Tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic men

In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the C with 5% CO[2] in 3% Bovine Serum spermatozoa were incubated up to 6h at 37 Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. The results upon western blotting showed: 1] at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2] The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished tyrosine phosphorylation efficiency in sperm from teratospermic men may be responsible for their compromised capacitation and low fertilization success rates