Evaluation of live birth rates and perinatal outcomes following two sequential vitrification/warming events at the zygote and blastocyst stages.

PURPOSE: To study the outcome of sequential cryopreservation-thawing of zygotes followed by the cryopreservation-thawing of blastocysts in the course of an IVF treatment on live birth rate and neonatal parameters. METHODS: Single center, retrospective chart review for the time period of 2015-2020. Clinical and perinatal outcomes were compared between frozen embryo transfer cycles utilizing twice-cryopreserved (n = 182) vs. once-cryopreserved (n = 282) embryos. Univariate and multivariable analyses were used to adjust for relevant confounders. RESULTS: After adjustment for maternal age, gravidity, parity, body mass index (BMI), paternal age, fertilization method used, the number of oocytes retrieved in the fresh cycle, fertilization rate, and transfer medium, the transfer of twice-cryopreserved embryos resulted in a reduced probability of live birth (OR, 0.52; 95% CI 0.27-0.97; p=0.041) compared to once-cryopreserved embryos. No differences in the sex ratio, the mean gestational age, the mean length at birth, or the mean birth weight were found between the two groups. CONCLUSION: The circumstantial use of sequential double vitrification-warming in course of treatment is associated with a reduced (but still reasonable) live birth rate compared to once-cryopreserved embryos. As the neonatal outcomes of twice-cryopreserved embryos are similar to once-cryopreserved embryos, this treatment option appears still valid as a rescue scenario in selected cases.

[Unaffected child born following preimplantation genetic diagnosis with karyomapping]. / Egészséges gyermek születése karyomapping eljárással történo preimplantációs genetikai diagnózist követoen.

Preimplantation genetic diagnosis for single gene defects is a well established method in assisted reproductive technologies. Karyomapping is a genome wide parental haplotyping using a high density single nucleotide polymorphism array that allows the diagnosis of any single gene defects. A couple with an affected child with primary congenital glaucoma attended at our clinic. Six oocyte-cumulus-complex was retrieved and all three mature oocytes were inseminated. One zygote showed the signs of normal fertilization and was cultured for five days. Trophectoderm biopsy and karyomapping analysis were carried out. Result showed a heterozygous carrier for primary congenital glaucoma. Embryo was thawed and transferred and a healthy girl was delivered at term. Here we report the first live birth following in vitro fertilization combined with preimplantation genetic diagnosis using karyomapping in Hungary. Karyomapping is able to accurately detect single gene disorders from a limited amount of samples without a significant preclinical workup. Orv. Hetil., 2016, 157(51), 2048-2050.

[Fertility preservation in female cancer patients.] / Daganatos nobetegek termékenységének megorzése.

The incidence of cancer increases with age and as family planning is being delayed, there is a growing number of cancer patients whose fertility may be affected by oncological treatments. International guidelines recommend that all reproductive age cancer patients, including adolescent patients, should be referred for fertility preservation consultation, and if necessary, fertility preservation procedures should be performed. Fertility preservation enables cancer survivors to offer a chance for biological parenthood after recovery. In this review, the gonadotoxic effects of oncological therapies and the fertility preservation possibilities for female cancer patients based on international recommendations and literature are discussed. Our next review will provide detailed information on the special fertility preservation possibilities for different cancer types. The two reviews may help to elaborate a national guidance. Orv Hetil. 2023; 164(28): 1094-1101.

Fertility preservation in cancer patients / A termékenység megorzése daganatos betegségekben. Egy hazai felmérés tapasztalatai

Introduction: Fertility preservation or oncofertility is a relatively new interdisciplinary field dealing with the preservation of female and male reproductive functions before the administration of gonadotoxic therapy. Despite recommendations from different international scientific bodies, Hungary still does not have a national fertility preservation network, patient referral is unorganised. Objective: As the first step towards establishing a national fertility preservation program, a study was designed to evaluate the Hungarian oncologists' knowledge, attitudes and practice in the field of oncofertility. Method: A national online survey was sent to the physician members of the Hungarian Society of Clinical Oncology between November 2020 and February 2021. The survey was completed by 94 physicians and the results were analysed statistically. Results: The majority of the oncologists (77%) discusses reproductive health issues before starting gonadotoxic therapy. However, almost half of these physicians do not refer patients for fertility preservation consultation or treatment. Physicians report lack of organised fertility preservation network, lack of knowledge and clinical practice guidelines as major barriers in referring their patients for fertility preservation. The majority (86%) proposes that a better col-laboration between cancer and fertility centers needs to be organized in Hungary. Conclusion: This study is the first nationwide survey to assess oncologists' attitude, knowledge and practice in the field of oncofertility in Hungary. It highlights the need for more education and increased collaboration between oncologists and reproductive specialists. This is an important step towards the establishment of a national fertility preservation network which is our ultimate goal.

Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos.

The effects of different activation methods and culture conditions on early development of porcine parthenotes were examined. Three different activation methods were tested: (1) electroporation; (2) electroporation followed by incubation in the presence of butyrolactone I, an inhibitor of cdc2 and cdk2 kinases; and (3) electroporation followed by a treatment with cycloheximide, a blocker of protein synthesis. The activated oocytes were cultured in two different media, NCSU-23 and PZM-3 under 5% CO2 in air. In a separate experiment, the effects of high (approximately 20%) or low (5%) O2 tension on early embryo development were also evaluated. The average pronuclear formation was less (p<0.05) in the electroporated oocytes (83.9+/-1.7%) compared with those activated by electroporation and butyrolactone I or electroporation plus cycloheximide (92.8+/-0.8 and 93.0+/-1.0%). In PZM-3 medium, the average frequencies of blastocyst formation (59.7+/-3.6%) and hatching (10.6+/-1.3%) were greater than those in NCSU-23 medium (39.9+/-3.1% blastocyst formation, p<0.05; and 0.2+/-0.2% hatching; p<0.001). Furthermore, the average nuclear number was also greater (p<0.001) in blastocysts developed in PZM-3 (50.2+/-1.3) than in those developed in NCSU-23 (35.3+/-1.1). Blastocyst formation was similar (p>0.10) among the three activation procedures when parthenotes were cultured in NCSU-23, while in PZM-3 more (p<0.05) parthenotes produced by electroporation plus butyrolactone or electroporation plus cycloheximide developed into blastocysts compared to electroporation alone (64.9+/-5.2 and 68.6+/-3.5% compared with 45.6+/-4.7%). Incidences of apoptotic nuclei were similar (p>0.10) among all treatments. No difference in development was found between parthenotes that developed under high versus low O2 tension (p>0.10). These results demonstrate that activation methods targeting the calcium signaling pathway at several points trigger embryonic development more efficiently than electroporation alone. The data also imply that the PZM-3 medium provides for enhanced culture conditions for the early development of parthenogenetic porcine embryos than NCSU-23.

Experiences in deer sperm cryopreservation under practical conditions--a pilot study.

The genetic potential of the red and fallow deer populations in Hungary is well known. Conserving the variability in this excellent genetic material for game preservation is one of our most important task. The aim of the present pilot study was to test the logistical steps of a sperm processing and storing system in which deer sperm can be stored at a level that meets quality standards accepted for domestic animals. Moreover, two different semen extenders, commercially used for freezing bull semen, were compared from the viewpoint of applicability to freeze fallow deer sperm. Sperm was collected from epididymes of eight red stags (Cervus elaphus hippelaphus) and six fallow bucks (Dama dama) during the rutting season. Red deer samples were washed in Triladyl extender, while fallow deer samples were split and processed in Triladyl or Bioxcell extender. In the samples, which had a shorter time interval between the death of the animal and the sperm collection, the percentage of viable spermatozoa with intact acrosome was typically higher.

Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination.

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17 and CREM were analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine 1/protamine 2 (P1/P2) ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects (2/20, 10%) showed an altered methylation pattern for the CREM gene promoter that was not found in control samples. These two samples had a significantly higher (P<0.05) promoter methylation (5.58 and 4.23%, respectively) compared to the control group (0.46%). In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREM gene, which may be either causative or a result of abnormal protmaine replacement.

Abnormal methylation of the promoter of CREM is broadly associated with male factor infertility and poor sperm quality but is improved in sperm selected by density gradient centrifugation.

OBJECTIVE: To evaluate the methylation status of the promoter of CREM in patients with an abnormal protamine 1/protamine 2 (P1/P2) ratio or oligozoospermia. The effects of density gradient centrifugation and aging of males on CREM promoter methylation were also investigated. DESIGN: Experimental research study. SETTING: University andrology and research laboratory. PATIENT(S): Patients with abnormally low (n=24) and high (n=36) P1/P2 ratio; patients with oligozoospermia (n=32); and normozoospermic, fertile controls with normal P1/P2 ratio (n=40). INTERVENTION(S): The CpG methyaltion pattern of the promoter of CREM was examined using pyrosequencing. MAIN OUTCOME MEASURE(S): The percentage of methylation of 12 CpGs in the CREM promoter was compared between patients and fertile controls. RESULT(S): There was a significantly higher rate of methylation of CREM in patients with abnormal protamination and oligozoospermia compared with the control group. Sperm concentration, sperm motility, and normal head morphology were negatively correlated with the amount of CpG methylation. Sperm selected from the 90% gradient fraction exhibited less methylation than sperm from the 35% fraction. CONCLUSION(S): Patients with two types of male factor infertility display an increased abnormal methylation of CREM compared with control subjects. Increased methylation is associated with decreased semen quality, and sperm selected by density gradient centrifugation have less methylation. Further research is necessary to investigate whether epimutations can be found on other nonimprinted gene promoters as well.

The clinical utility of the protamine 1/protamine 2 ratio in sperm.

During spermiogenesis, human sperm undergo a dramatic reorganization of the chromatin in which canonical histones are replaced by two types of protamines, protamine 1 (P1) and protamine (P2). P1 and P2 are expressed approximately at a 1:1 ratio in healthy men. Alteration of this ratio is associated with male infertility. Patients with an abnormal P1/P2 ratio generally exhibit diminished semen quality, lower fertilization ability, and lower pregnancy rates when undergoing in vitro fertilization. Many studies have reported an elevated incidence of abnormal P1/P2 ratios in infertile men compared to fertile controls, and have evaluated the relationship between infertility and abnormal protamination; however, no prospective study has investigated the normal range of the P1/P2 ratio in men from the general population. Here, we report a P1/P2 reference range of 0.54 to 1.43 in a fertile, normozoospermic population. This rather wide normal range of P1/P2 led us to the conclusion that abnormal protamination is more likely indicative of other perturbations during spermatogenesis than the underlying mechanism to cause infertility. Alternatively, protamine expression may act as a checkpoint mechanism and thus be indirectly related to semen quality.

The marmoset and cotton rat as animal models for the study of sperm chromatin packaging.

In the human, canonical histones are largely replaced by protamine 1 (P1) and protamine 2 (P2) during late spermatogenesis. Recent studies have demonstrated that abnormal replacement of the histones is associated with severe male infertility and has profound implications for early embryogenesis. In this review the hispid cotton rat and the common marmoset are evaluated as animal models for the study of chromatin packaging in sperm. The DNA sequences of protamine 1 and protamine 2 in the cotton rat are also reported. We have found that the P1 and P2 genes share a 64% and 56% amino acid identity with human, respectively. The hispid cotton rat expresses both protamines, has a similar P1:P2 ratio, and a gene sequence homologous to human, thus making it a possible model organism for chromatin studies. The common marmoset also expresses both protamine genes and has a very similar spermatogenetic characteristic to man. This review considers both animals as possible models to study the mechanisms of protamine regulation and the effects of altered expression caused by environmental or physiological perturbations.

Comparison of 5% and ambient oxygen during days 3-5 of in vitro culture of human embryos.

OBJECTIVE: To compare the effect of two oxygen concentrations used during days 3-5 of human embryo culture on embryo quality and pregnancy outcome. DESIGN: Retrospective analysis of the use of two culture conditions. SETTING: University-based infertility clinic. PATIENT(S): Three hundred eighty-two patients undergoing IVF. INTERVENTION(S): Embryos were cultured in 5% CO(2) balanced ( approximately 20% O(2)) gas phase until day 3 then assigned to approximately 20% or reduced (5%) oxygen concentration groups and cultured until ET. MAIN OUTCOME MEASURE(S): Embryo quality, pregnancy rates, and implantation rates. RESULT(S): There were no differences in demographic features (age, type of infertility) between the two groups. The embryo scores at day 3 and day 5, blastulation rate, and transfer score did not differ between groups. No differences were observed between the 5% and 20% oxygen concentrations in the chemical pregnancy rate (71.27% vs. 78.72%), clinical pregnancy rate (58.56% vs. 64.36%), or implantation rate (44.06% vs. 44.16%). CONCLUSION(S): Reduced oxygen concentration in the gas mixture from day 3 until ET did not support better embryo development or result in higher pregnancy or implantation rates. These data do not support the hypothesis that beneficial effects of reduced oxygen concentration can be gained by employing the strategy during the latter stages of embryo culture (days 3-5) only and highlight the need for further studies through all stages of in vitro culture.

Incidence of apoptosis in parthenogenetic porcine embryos generated by using protein kinase or protein synthesis inhibitors.

In mammals, embryonic development can be artificially initiated by activating the oocyte using a number of methods. In the present research, we investigated whether butyrolactone I and cycloheximide, two chemicals frequently used in combined oocyte activation protocols, have any detrimental effect on programmed cell death in the developing porcine embryo. Parthenogenetic porcine blastocysts were generated by the following methods: (1) electroporation followed by blocking the activity of specific protein kinases with butyrolactone I; (2) electroporation followed by inhibition of protein synthesis using cycloheximide; and (3) electroporation only (control). The viability of the embryos was evaluated by monitoring the frequency of cells with early and late signs of programmed cell death. There was no difference between the embryos in terms of blastocyst formation (ranging from 27.5+/-3.8 and 33.3+/-3.5%) and total nuclear number (ranging from 23.5+/-1.1 and 31.8+/-4.4). In addition, the occurrence of apoptosis was also similar in the three experimental groups. The proportion of cells with active caspase-9 (a sign of early apoptosis) in the blastocysts produced by the different activation methods was between 19.4+/-1.9 and 23.0+/-2.4%. The annexin V assay revealed that phosphatidylserine flip (another early apoptotic event) took place in 24.4+/-2.1 to 32.3+/-2.4% of the blastomeres. Finally DNA fragmentation, a sign of late stage apoptosis determined by the TUNEL assay, occurred in 8.5+/-2.4 to 10.1+/-3.0% of the cells. The results indicate that temporary inhibition of specific protein kinases or protein synthesis does not increase the onset of apoptosis in parthenogenetic porcine blastocysts.

Paternal effects on early embryogenesis.

Historically, less attention has been paid to paternal effects on early embryogenesis than maternal effects. However, it is now apparent that certain male factor infertility phenotypes are associated with increased DNA fragmentation and/or chromosome aneuploidies that may compromise early embryonic development. In addition, there is a growing body of evidence that the fertilizing sperm has more function than just carrying an intact, haploid genome. The paternally inherited centrosome is essential for normal fertilization, and the success of higher order chromatin packaging may impact embryogenesis. Epigenetic modifications of sperm chromatin may contribute to the reprogramming of the genome, and sperm delivered mRNA has also been hythesized to be necessary for embryogenesis. There is less information about the epigenetic factors affecting embryogenesis than genetic factors, but the epigenetics of gamete and early embryogenesis is a rapidly advancing field.