Comparative analysis of conventional in vitro fertilization and intracytoplasmic sperm injection in patients with polycystic ovarian syndrome, tubal factor infertility, and unexplained infertility whose partners exhibit normal semen parameters: a retrospective study of sibling oocytes.

Objective: This study compared the outcomes of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in patients with polycystic ovarian syndrome (PCOS), tubal factor (TF) infertility, and unexplained infertility whose partners had normal semen parameters. Methods: This retrospective study included 360 couples diagnosed with infertility involving PCOS (n=157), unexplained infertility (n=140), and TF infertility (n=63). Sibling oocytes were randomly assigned to undergo ICSI or conventional IVF insemination. The fertilization rate and embryo morphology were evaluated as outcomes. Results: Retrieved cumulus-oocyte complexes from patients with PCOS (2,974), unexplained infertility (1,843), and TF infertility (844) were split and inseminated by conventional IVF and ICSI respectively. In comparison to the ICSI method, the conventional IVF approach was linked to a significantly higher fertilization rate in groups with PCOS (68.81% vs. 77.49%), unexplained infertility (67.62% vs. 78.84%), and TF issues (69.23% vs. 78.63%) (p<0.05). The proportion of embryos with grade A produced by the conventional IVF method was significantly higher than that produced using the ICSI method in the PCOS and unexplained infertility groups (p<0.05). Additionally, the percentage of grade B embryos produced with the ICSI method was significantly higher than that produced with the conventional IVF method in PCOS patients (p=0.002). Conclusion: Our results indicated that the conventional IVF method was associated with higher zygote production and a higher proportion of grade A embryos when all infertile groups were evaluated together. Thus, ICSI is not suggested for patients with these causes of infertility if their partner has normal semen parameters.

Implication of Novel BMP15 and GDF9 Variants in Unexpected Poor Ovarian Response.

Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27 IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744 T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.

Comparing the effects of vitrification, before and after mouse oocyte in vitro maturation on developmental competence, changes in epigenetic regulators and stress oxidative response.

Since the developmental stage of oocyte is a challenging issue in the success of vitrification, this study investigated the effects of vitrification, before and after in vitro maturation, on the survival and maturation rates, developmental competence and the expression levels of genes involved in apoptosis, oxidative stress and epigenetic modifications. Mouse germinal vesicle (GV) oocytes were divided into four groups: fresh in vitro matured oocytes without vitrification (fIVM), in vitro matured oocytes after vitrification (vIVM), in vitro matured oocytes before vitrification (IVMv). In addition, in vivo matured oocytes (MII) were used as control. After oocytes collection, maturation and survival rates as well as the intracellular reactive oxygen species (ROS) level were evaluated. Also, the expression level of various genes was analyzed by qRT-PCR. In addition, following artificial activation (parthenogenesis), the developmental competence of oocytes to the blastocyst stage was evaluated. A significant decrease in maturation rate and survival of vIVM oocytes was observed compared to fIVM and IVMv oocytes. Intracellular ROS levels were significantly increased in both vitrified groups compared to the fIVM group, and no significant difference between vitrified groups. Pro-apoptotic genes; BAX and Bcl2 as well as genes related to oxidative stress response Hsp1a, Hsp1b and SOD1were significantly increased in the vIVM group compared to the IVMv group. Interestingly, epigenetic regulators genes DNMT1, DNMT3a and DNMT3b were highly expressed in IVMv oocytes along with a decrease in the artificial activation rate compared to the vIVM oocytes. Our results indicated that despite observing more negative effects of vitrification before IVM on the survival rate and maturation as well as apoptosis status, less epigenetic changes in vIVM oocytes can make this process a better option in the treatment of infertility than IVM of oocytes followed by vitrification, a hypothesis that needs to be investigation in human oocytes.

Impact of oxidative stress SNPs on sperm DNA damage and male infertility in a south-east Iranian population.

AIM: We examined four single nucleotide polymorphisms in four antioxidant genes (PON1 , CAT , GPx1 and SOD2 ) in 100 infertility cases and 100 controls from an Iranian population-based case-control study to confirm the assumption that polymorphisms in oxidative stress genes increase the risk of sperm DNA damage and idiopathic male infertility. METHODS: Restriction fragment length polymorphism and tetra-primer amplification refractory mutation system PCR were used to identify genotypes. Sperm DNA damage was assessed using the Sperm Chromatin Dispersion test (Halo Sperm), and the total antioxidant capacity of seminal fluid was determined using the FRAP assay. KEY RESULTS: Our findings demonstrated that alleles Arg-PON1 (rs662) and Ala-MnSOD (rs4880) variant genotypes were considerably linked with a higher risk of male infertility. CONCLUSIONS: Linear regression analysis revealed that those with the PON1 Gln192Arg or SOD2 Val16Ala variants have significantly higher levels of sperm DNA fragmentation and lower levels of the total antioxidant capacity in seminal fluid. IMPLICATIONS: These findings suggest that genetic differences in antioxidant genes may be linked to oxidative stress, sperm DNA damage, and idiopathic male infertility.

Successful approach for couples having their own genetic offspring using vitrified-warmed oocytes injected by frozen-thawed poor quality testicular sperm: A case report.

The clinical applications of donated gametes are approved in many countries; however, attitudes toward its application and national legislation in some countries are challenging. The purpose of this study is to report a healthy live birth produced by vitrified-warmed oocytes and frozen-thawed testicular sperms to avoid sperm donation.

Effects of human chorionic gonadotropin intrauterine injection on oocyte retrieval day on assisted reproductive techniques outcomes: An RCT.

BACKGROUND: Several mediators play an important role in implantation. One of these mediators is human chorionic gonadotropin (HCG). OBJECTIVE: To evaluate the effects of HCG intrauterine injection on the day of oocyte retrieval on the result of assisted reproductive techniques (ART). MATERIALS AND METHODS: In this randomized clinical trial study, 126 women who were referred to Afzalipour Infertility Center between December 2018 to December 2019 undergoing in vitro fertilization/intracytoplasmic sperm injection cycles were enrolled and assigned to two groups of: a case (n = 62) and a control group (n = 64). The protocols for both groups were the same; except that the case group was injected with the protocols for both groups were the same, except that the case group was injected with 1000 IU of HCG into uterine cavity following the oocyte puncture, while no medication was administered to the control group. The implantation rate, chemical pregnancy, clinical pregnancy, and abortion rates were compared between the two groups. RESULTS: Positive chemical pregnancy was seen in 15 (27.3%) cases of the case group and 14 (25.5%) of the control group. No significant difference was seen in the chemical and clinical pregnancy rates between the groups. The abortion rate was higher in the control group but that was not significant. CONCLUSION: A 1000 IU of HCG intrauterine injection after oocyte retrieval does not improve implantation, chemical or clinical pregnancy rates in ART cycles. Further studies are needed to clearly understand the role of HCG intrauterine injection in the day of oocyte retrieval in ART outcomes.

Bacteriospermia and its association with seminal fluid parameters and infertility in infertile men, Kerman, Iran: A cross-sectional study.

Background: The role of genital Ureaplasma species, genital Mycoplasma (M) species, and Chlamydia (C.) trachomatis, the most prevalent sexually transmitted bacteria, in male infertility are still not clear. Different reports about the impact of these bacteria on semen quality are controversial. Objective: This study was proposed to determine the frequency of bacteriospermia in men and investigate the relationship between the presence of these bacteria and semen quality using molecular assay. Materials and Methods: In this cross-sectional study, 200 semen samples obtained from men attending the research and clinical centers for fertility in Kerman, Iran, between July and December 2019 were analyzed for semen volume, progressive motility, non-progressive motility, total progressive motility, and viability according to the World Health Organization guidelines. The polymerase chain reaction was used for the detection of related bacteria. Results: The mean values of volume, progressive motility, non-progressive motility, total progressive motility, and viability were significantly lower in infertile men (p < 0.001). Statistically significant correlations were observed between the presence of M. genitalium and progressive sperm motility, M. hominis and semen volume, Ureaplasma parvum and the sperm normal form, and C. trachomatis and the sperm progressive motility and viability. Logistic regression analysis showed that M. genitalium (OR = 8.06, p < 0.001) and C. trachomatis (OR = 16, p = 0.01) were significantly associated with male infertility. Conclusion: During the infertility assessment, clinicians should consider of role C. trachomatis and M. genitalium in male infertility. Screening test particularly for asymptomatic individuals is recommended.

Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts.

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts. METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined. RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05). CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

Does rescue in vitro maturation of germinal vesicle stage oocytes impair embryo morphokinetics development?

SummaryCurrently, rescue in vitro maturation (IVM) is not a routine method in assisted reproductive treatment (ART) programmes but is a promising procedure for ART to improve IVM. The aim of this study was to compare embryo morphokinetics of germinal vesicles (GV) with metaphase II (MII) oocytes from controlled ovarian hyperstimulation (COH) cycles by time-lapse photography monitoring (TLM). Morphokinetics of the same number of embryos derived from the in vivo (group I) and rescue of in vitro matured oocytes (group II) from 310 patients were analyzed and compared retrospectively. The time to form second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf) and time of two to eight discrete cells (t2-t8) were assessed. Abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), trichotomous mitoses (TM), and the rates of embryo arrest were assessed. These data showed that tPB2, tPNa, tPNf, t2, t3 and t4 stages took place later in group II compared with group I (P<0.001, P=0.017, P<0.001, P<0.001, P<0.001, P<0.001, respectively). The rates of uneven blastomeres, Fu, TM, and embryo arrest were increased significantly in group II compared with group I (P=0.001, P<0.001, P=0.003, P<0.001, respectively). Based on the exact annotation of timing parameters and cleavage patterns, the present data agreed with the concept that rescue IVM of oocytes negatively influences embryo morphokinetics. Therefore, cautious use of embryos derived from rescue IVM of GV oocytes should be made.

Vitrification of human immature oocytes before and after in vitro maturation: a review.

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

Oocyte morphology and embryo morphokinetics in an intra-cytoplasmic sperm injection programme. Is there a relationship?

The aim was to investigate the relationship between the morphological parameters of metaphase II (MII) oocytes with morphokinetic variables of embryos following an intra-cytoplasmic sperm injection (ICSI) procedure. Morphokinetic behaviour and abnormal cleavage patterns of 334 zygotes were analyzed using time-lapse monitoring (TLM). In addition, oocyte morphology was assessed in relation to embryo morphokinetic (absolute time point, including time to second polar body (PB) extrusion (ESPB), pronuclei (PN) appearance (PNA), PN fading (PNF), time to 2-cells (t2), 3c (t3), 4c (t4), 5c (t5), 6c (t6), 7c (t7), 8c (t8) and relative timing parameters (S1, S2, CC2 and CC3). Also, cleavage patterns (uneven blastomeres, reverse, direct and arbitrary) were assessed. The data showed that 79% of the normal fertilized oocytes had at least one abnormal morphological characteristic. Intra-cytoplasmic abnormalities were observed in 12% of the oocytes. Also, extra-cytoplasmic abnormalities were noticed in 29%, while combined intra- and extra-cytoplasmic abnormalities were responsible for the remaining 38% of the oocytes. Nearly all cleavage and interval times, except extrusion of the ESPB time (P = 0.003), were similar between normal and abnormal morphologic oocytes (P < 0.05). Moreover, there was significant relationship for oocyte morphology abnormalities and cleavage patterns, including uneven blastomere (P = 0.037), reverse cleavage (RC) (P = 0.0), direct (P = 0.001) and arbitrary cleavages (P = 0.001). Using TLM, the cleavage patterns of embryos were affected by the quality of MII oocytes in the ICSI cycles. So, evaluation of oocyte morphology with subsequent embryo morphokinetics is recommended in assisted reproductive programmes.

Analysis of meiotic spindle and zona pellucida birefringenceof IVM oocytes in PCOS patients.

BACKGROUND/AIM: Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility. One of the best therapeutic options for PCOS patients is intracytoplasmic sperm injection (ICSI). In vitro maturation (IVM) can also be a useful technique for these women. The goal of this study was to evaluate both the zona pellucida (ZP) birefringence and meiotic spindle (MS) of in vivo- and in vitro-matured oocytes from PCOS patients using the PolScope system. MATERIALS AND METHODS: Immature oocytes undergoing IVM and MII oocytes were obtained from PCOS patients in an ICSI program. Using PolScope, the presence of MS and ZP birefringence was assessed in both in vivo-matured oocytes (n = 32) and IVM oocytes (n = 24). Oocytes were classified as having highly birefringent (HB) ZP and lowly birefringent (LB) ZP. Furthermore, the rates of fertilization after ICSI were evaluated. RESULTS: The maturation rate was 68.5% after IVM. The percentage of a HB-ZP was significantly higher in the IVM oocytes than in vivo-matured ones (58.3% vs. 31.2%, respectively; P = 0.04). There were similar outcomes for the fertilization rates and MS detection between the two groups (P = 0.80 and P = 0.53, respectively). CONCLUSION: Clinical IVM is a safe technology for the maturation and maintenance of oocyte integrity in PCOS patients. The use of the noninvasive PolScope is recommended for detection of healthy oocytes in ICSI.

Sperm parameters, protamine deficiency, and apoptosis in total globozoospermia.

BACKGROUND: Globozoospermia is a severe form of teratozoospermia (incidence < 0.1%) in infertile men that is characterized by round headed sperm and acrosomeless in semen. OBJECTIVE: To compare the semen parameters, protamine deficiency, and apoptosis in ejaculated spermatozoa between globozoospermic and normozoospermic men. MATERIALS AND METHODS: Thirty six semen samples were divided into two groups including 15 infertile men with total globozoospermic (> 90% round-headed sperm) and 21 healthy donors with normal spermograms as controls. Semen analysis was performed according to World Health Organization criteria (2010). Sperm protamine deficiency was assessed using Chromomycin A3 (CMA3) staining and the rate of apoptotic spermatozoa was evaluated with TUNEL assay. RESULTS: Sperm concentration, motility, and normal morphology in globozoospermic men were significantly decreased compared with controls (p<0.05). The rate of CMA3-reacted spermatozoa (CMA3+) in globozoospermic men was higher than controls (65.93 ± 11.77 vs. 21.24 ± 7.37, respectively, p<0.0001). The rate of apoptotic spermatozoa (TUNEL positive) were significantly increased in globozoospermic cases with respect to the controls (17.60 ± 10.72 and 5.95 ± 3.02, respectively, p<0.0001). There was no significant correlation between sperm protamine deficiency and apoptosis in globozoospermic men. CONCLUSION: Globozoospermic samples contain a higher proportion of spermatozoa with abnormal chromatin packaging and DNA fragmentation than normozoospermic samples. Therefore, in addition to absence of acrosome in the spermatozoa of globozoospermic patients, the high percentage of spermatozoa with immature chromatin and apoptotic marker may be considered as the other etiologies of infertility in these patients.

Developmental competence of immature oocytes aspirated from antral follicles in patients with gynecological diseases.

BACKGROUND: In vitro maturation (IVM) of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, quality of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive Polscope system, both meiotic spindle (MS) and zona pellucida (ZP) can be assessed in living oocytes. OBJECTIVE: The aim was to investigate the developmental potential of immature oocytes retrieved from ovarian tissue after IVM, as a method for fertility preservation, in patients with gynecological diseases. MATERIALS AND METHODS: The ovarian cortex from 26 patients with malignant and benign diseases (21-45 years old), were obtained directly from collaborating hospitals, and transported to the IVF center on ice. In total 61 immature oocytes were aspirated, of which 18 (29.5%) were degenerated and discarded. The remaining 43 (70.5%) healthy oocytes were cultured in IVM culture media for 48 hr. The rate of maturity was assessed, and the ZP birefringence and MS were imaged with Polscope technology. RESULTS: Overall 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II (MII) oocytes. The ovarian tissues of 9 (34.6%) women were lacking oocytes at any stage. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringence's were observed in the majority of the oocytes post IVM (61.5%). CONCLUSION: Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in gynecologic patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation.

Noninvasive assays of in vitro matured human oocytes showed insignificant correlation with fertilization and embryo development.

PURPOSE: Recently, the upgrading of in vitro maturation (IVM) of human oocytes as a promising strategy has emerged in assisted reproductive technology (ART). The goal was to evaluate the correlation of the in vitro matured oocytes selected on the basis of the zona pellucida (ZP) birefringence and meiotic spindles (MS) detection with fertilization and subsequent embryo development in ICSI program. METHODS: A total of 168 immature oocytes [germinal vesicle (n = 140) and metaphase I (n = 28)] obtained from patients undergoing oocytes retrieval for ICSI. After in vitro culture for 24-40 h, 112 (67 %) oocytes reached to MII stage. Using a polarized microscopy, the presence of MS and ZP birefringence were assessed in matured oocytes, followed by ICSI performance. RESULTS: The rates of fertilization in oocytes with spindles (51.3 %) were similar to that of the oocytes without spindles (50.7 %; P = 1.00). Moreover, the fertilization rates in high birefringence (HB) oocytes was not statistically different than oocytes with low birefringence (LB) (P = 0.44). The findings also showed that 64.9 % of the fertilized oocytes developed to embryos, in which 33.3 % were derived from spindle-detected oocytes. Regarding the ZP birefringence, 35.5 % of the embryos were derived from HB oocytes. CONCLUSIONS: There were insignificant relationships between the MS detection and ZP birefringence score with the rates of fertilization and embryo development in IVM oocytes.

Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding.

Human sperm vitrification is a new cryopreservation method. This study compared the effects of rapid freezing and vitrification on various sperm parameters, hyaluronan-binding assay and DNA fragmentation and assessed the impact of cryoprotectant agents (CPA) with vitrification. A total of 30 normo-ejaculates were prepared by swim up and the motile sperm fraction was divided into four: fresh (control), rapid freezing, and two vitrification groups (a, lacking CPA; b, with CPA). For rapid freezing, a cryovial of sperm suspension was held just above the liquid nitrogen surface, and for vitrification, 30µl suspension was dropped directly into liquid nitrogen. Sperm parameters, including motility, viability and morphology, declined after cryopreservation in both groups. DNA fragmentation was not significantly higher in the vitrification (15.7±4.4%) or rapid freezing (16.6±5.6%) groups when compared with controls (11.6±4.5%). The rates of hyaluronan binding were similar between the control and cryopreserved groups. Moreover, addition of CPA for vitrification had a neutral effect on rates of sperm recovery. In conclusion, vitrification has great potential for human sperm cryopreservation and does not require CPA, with its possible toxicity. However, it is not superior to rapid cryopreservation regarding sperm recovery rate in normozoospermia. Human sperm vitrification is a new cryopreservation method that has been introduced recently. This study compared the effects of rapid freezing with vitrification on rates of sperm parameters, hyaluronan-binding assay and DNA fragmentation after thawing/warming and assessed the impact of cryoprotectant agent (CPA) on vitrification. The study was performed on 30 ejaculates prepared using the swim-up technique. Each motile sperm suspension was divided into four: control (fresh); rapid freezing; and two vitrification groups (a, lacking CPA; b, with CPA). For rapid freezing, a cryovial of sperm suspension was held above the surface of liquid nitrogen. For vitrification, 30µl sperm suspension was dropped into liquid nitrogen directly. The rates of progressive motility (86.6±5.9%) and viability (95.8±3.9%) in controls declined significantly, to 40.0±13.0% and 63.2±7.7% for rapid freezing and 41.9±10.3% and 64.4±10.0% for vitrification, respectively. Normal sperm morphology was also significantly decreased after cryopreservation in all groups. DNA fragmentation was higher with rapid freezing compared with fresh controls (16.6±5.6% vs. 11.6±4.5%, P=0.01), but DNA fragmentation did not increase significantly in vitrified samples (15.7±4.4%). The rates of hyaluronan binding were similar between the control and cryopreserved groups. Moreover, addition of CPA for vitrification had a neutral effect on rates of sperm recovery. In conclusion, vitrification has great potential for human sperm cryopreservation and does not require CPA, with its possible toxicity. However, it is not superior to rapid cryopreservation regarding sperm recovery rate in normozoospermia.

Zona pellucida birefringence and meiotic spindle visualisation of human oocytes are not influenced by IVM technology.

The aim of the present study was to investigate the relationship between the presence of the meiotic spindle and zona pellucida (ZP) birefringence with morphology of in vivo- and in vitro-matured human oocytes. Germinal vesicles (n=47) and MI (n=38) oocytes obtained from stimulated ovaries of patients undergoing intracytoplasmic sperm injection (ICSI) underwent IVM. Using a PolScope (OCTAX PolarAID; Octax, Herbon, Germany), the presence of spindles and ZP birefringence was assessed in both in vivo-matured (n=56) and IVM (n=56) oocytes. In addition, the morphology of each matured oocyte was evaluated microscopically. There were insignificant differences for ZP birefringence and meiotic spindle between the in vivo-matured and IVM MII oocytes. Subanalysis revealed that the rates of morphologically abnormal oocytes did not differ significantly between the two groups, except in the case of irregular shape (P=0.001), refractile body (P=0.001) and fragmented polar body (P=0.03), which were higher in IVM oocytes. In the case of in vivo-matured oocytes, a significantly higher percentage of oocytes with intracytoplasmic and both intra- and extracytoplasmic abnormalities have a low birefringent ZP (P=0.007 and P=0.02, respectively). There was no relationship between morphological abnormalities and spindle detection. The findings suggest that clinical IVM is a safe technology that maintains the high maturation rate and integrity of oocytes. In addition, the use of the non-invasive PolScope is recommended for the detection of oocytes most suitable for ICSI.

Does women's age influence zona pellucida birefringence of metaphase ΙΙ oocytes in in-vitro maturation program?

BACKGROUND: In vitro maturation (IVM) is a promising treatment option for certain infertile women. Nowadays, with the aid of PolScope, it has become possible to evaluate zona pellucida (ZP) characteristics as a parameter of oocyte quality. Moreover, quality of oocytes can be influenced by many factors, such as patient's age. The PolScope system is a non-invasive technique to assess birefringent structures such as the meiotic spindle and ZP in living oocytes. OBJECTIVE: The aim was to determine the influence of the woman's age on ZP birefringence, a sign of oocyte quality, and morphology of in-vitro matured human oocytes using non-invasive polarized light (PolScope) microscopy. MATERIALS AND METHODS: ZP birefringence and morphology were determined in 105 retrieved oocytes from 58 women undergoing ICSI in two age groups (≥30 years and <30 years). The immature oocytes were selected and after IVM, the quality of metaphase ΙΙ (MII) oocytes was assessed. The oocytes abnormalities were classified as intracytoplasmic and extracytoplasmic abnormalities. RESULTS: Oocyte maturation rates were significantly reduced in ≥30 year's women (56%) in comparison with other age group (80.7%). In addition, the ZP birefringence was significantly higher in MII oocytes in the younger group compared with the older group (76.2% vs. 38.1%; p=0.00). Following morphologic assessment, the rates of oocytes with extracytoplasmic (p=0.02) and both abnormalities (extra- and intracytoplasmic) (p=0.01) were higher in aged versus the younger women. CONCLUSION: There was a positive relationship between advanced maternal age with decreased ZP birefringence and oocyte morphological quality in in-vitro matured human oocytes.