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PURPOSE: Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization. METHODS: Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2–3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST. RESULTS: For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2–3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population. CONCLUSION: Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-022-02401-7.

PURPOSE: To examine the intra- and inter-individual variability in fatty acid composition of follicular fluid (FF) of 23 patients undergoing assisted reproductive treatment. METHODS: The average coefficient of variation within each patient (CV(w)) and intra-class correlation coefficient (ICC) values of FF fatty acid composition as well as correlation between the fatty acid composition of individual, pooled or first-punctured follicles, were assessed. RESULTS: The proportions of 16:0, 18:0, cis-9 18:1, 18:2n-6, 20:5n-3, total MUFA and n-3 PUFA showed good reproducibility (CV(w)

Purpose The importance of the surrounding ovarian stromal cells and extracellular matrix in the development and maturation of follicles has recently gained attention. An aberrant extracellular matrix has been described in ovaries of patients with polycystic ovary syndrome where a more rigid structural environment, possibly induced by endogenous testosterone, impairs normal folliculogenesis. In this context, we describe the textural parameters of the ovarian cortex of transgender men after prolonged testosterone administration compared to the textural parameters of the non-exposed ovarian cortex originating from female oncological patients. Methods Texture profile analysis (TPA) was performed on ovarian cortex (5 × 5 mm) of oncological and transgender patients in order to measure stiffness, hardness, cohesiveness, and springiness of the ovarian cortex (LRXplus universal testing system). Statistical analysis was performed using repeated measurements mixed models and the Spearman rank order correlation test (IBM SPSS Statistics 23). Results A total of 36 frozen-thawed cortical strips (5 × 5 mm) were subjected to TPA. The superficial part of cortex fragments originating from transgender persons (fragments < 1.4 mm; N = 10) appeared to be significantly stiffer compared to cortex derived from oncology patients (fragments < 1.4 mm; N = 7) (6.78 ± 1.38 N/mm versus 5.41 ± 0.9 N/mm respectively, p = 0.036). Conclusions This is the first application of TPA in ovarian cortex to study the physical properties. Comparing the physical properties, we objectively describe an increased cortical stiffness in the most outer part of the ovarian cortex following prolonged testosterone administration in transgender men compared to the ovarian cortex of oncological patients. This preliminary and novel approach could be the start of future research to understand the physical properties of ovarian tissue. Electronic supplementary material The online version of this article (10.1007/s10815-019-01513-x) contains supplementary material, which is available to authorized users.

Purpose: A matched case–control study of all pregnancies obtained after either IVF or ICSI was conducted to investigate the perinatal outcome. Methods: Three hundred eleven singleton and 115 twin pregnancies obtained after assisted reproduction were studied. Controls were selected from a regional register and were matched for maternal age, parity, singleton or twin pregnancy, and date of delivery. Results: No significant difference was observed for gestational age at delivery, birth weight, incidence of congenital anomalies, and incidence of perinatal mortality between ART (singleton and twin) pregnancies and spontaneous controls. ART twin pregnancies showed a higher incidence of preterm deliveries than control pregnancies (52 vs 42%; P < 0.05) and needed more neonatal intensive care (47 vs 26%; P < 0.05). Conclusions: From this case–control study it is concluded that the perinatal outcome of ART singleton pregnancies is not different from that in matched controls. ART twin pregnancies showed a higher incidence of preterm deliveries than control pregnancies and needed more neonatal intensive care.

The objective of this study was to evaluate the efficiency of vitrified blastocysts derived from frozen-thawed cleavage stage embryos in terms of morphological survival and re-expansion status of the blastocoelic cavity.After warming 162 blastocysts derived from fresh embryos (= control group) and 90 blastocysts from frozen-thawed cleavage stage embryos (= study group) and after 2-3 h of in vitro culture the percentage of blastocysts with morphological survival was not different between the two groups. After 24 h of in vitro culture, the percentage of fully expanded, hatching or hatched blastocysts was not different between both groups.The results show that blastocysts derived from frozen-thawed cleavage stage embryos can be cryopreserved successfully a second time by vitrification method. Re-cryopreservation by vitrification still needs to be approached with some caution because little data on long term safety of multiple freezing is available.

Our objective was to develop a new reliable method for cytogenetic analysis of the chromosome set in second polar bodies (PBs) from one-cell-stage mouse embryos.The study took place at the Reproductive Biology and Experimental Cytogenetics Laboratories.Oocytes from F1 hybrid and T6/T6 mice were fertilized in vitro and artificially activated with ethanol. Zygotes, parthenogenetic embryos, and isolated second PBs were treated with 10 microM okadaic acid (OA) for 1-2 hr, further cultured in plain medium, and fixed. Chromosomal preparations were made and C-banded, and the number of chromosomes in second PBs and embryos was counted.OA-induced nuclear envelope breakdown in pronuclei as well as in second PB nuclei. Countable chromosome plates were obtained in 92-93% of second PBs treated 4-4.5 hr after activation. The T6 marker chromosome could easily be recognized in second PBs from T6/T6 mice. A haploid set of chromosomes was obtained in 18 of 19 isolated second PBs treated with OA 4-5 hr after activation.Treatment of second PBs with OA allows visualization of the PB chromosomes. Cytogenetic analysis of the second PB and the corresponding oocyte constitutes a new approach for the study of meiotic nondisjunction in experimental cytogenetics. The chromosomal study of isolated second PBs seems to be promising for clinical preimplantation cytogenetics.

Erratum to: J Assist Reprod Genet (2015) DOI 10.1007/s10815-015-0496-0 The original version of this article unfortunately contained a mistake. The family name of Bjorn Heindryckx was misspelled, omitting letter “x”. The update is provided here as well.

Purpose: The case history of a 36-year-old patient with transient ovarian failure, presumably induced by previous ovarian surgery and a recent pelvic inflammatory disease, is described. Methods: During several months, the patient underwent transvaginal ultrasound and hormonal follow-up. Ovulation was triggered by hCG in a cycle in which spontaneous follicular ripening had occurred. Transvaginal puncture of the single follicle was performed for in vitro fertilization. Results: One oocyte was retrieved and fertilized by intracytoplasmic sperm injection, giving rise to a single goodquality embryo. A singleton pregnancy, which ended in the birth of a healthy daughter, was established. Conclusions: This case demonstrates that it is worthwhile to monitor patients with transient ovarian failure closely, and if other infertility factors are present, IVF of one oocyte may be considered.

Transport in vitro fertilization (IVF) programs are operational in a lot of countries and especially popular in The Netherlands, where IVF activities are strictly regulated. Since the introduction of intracytoplasmic sperm injection (ICSI) in the IVF laboratory, many laboratories are now setting up this new technique, which necessitates major investments in terms of infrastructure and specialized personnel.We present a cost effective alternative, consisting of patient selection, preparation, and oocyte retrieval at one center and transport of oocytes to a second center, where the ICSI procedure and embryo transfer are performed. Since early 1994 several Dutch centers have a transport ICSI program running with the Gent University Infertility Center, and we wish to present the results of our cooperation with two major centers, comparing them to our local results, for the first 10 months of 1994. Patient selection was similar at all three centers: only couples with previously failed in vitro fertilization or having been refused for routine IVF were enrolled in the program. Stimulation schemes and follow-up of the stimulation were different at all three centers. Transport of oocytes was carried out in a transport box or by attaching the closed tubes containing the follicular aspirates to the chest of the husband. Transport times varied between 1.5 and 3 hr, depending on traffic conditions.Up to November 1, 1994, a total of 77 transport ICSI cycles and 294 own ICSI cycles were carried out. Although locally significantly more oocytes were retrieved and thus available for ICSI than in transport cycles, fertilization and pregnancy rates were not different between the two groups.These results suggest that long-distance transport of human oocytes seems not to be harmful to their capacity to be successfully injected and to further embryonic development and their implantation potential. Transport ICSI seems to be a valuable and cost-effective approach to treat high numbers of patients at a restricted number of highly specialized IVF laboratories, especially in countries where ICSI is not commonly available.

Purpose: Preliminary data from some research centers indicate that assisted hatching might be of value to increase embryo implantation rate in the human, at least in selected cases. It is not clear, however, whether this technique would be of benefit for all patients undergoing an embryo transfer. We therefore performed a prospective randomized study to evaluate the effect of assisted hatching on the implantation rate in our in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) program. Methods: In total, 120 couples undergoing an embryo transfer were randomized between two groups: in one group no assisted hatching was performed (AH−), whereas in the other group the embryos selected for transfer were subjected to partial zona dissection (PZD) immediately prior to the transfer (AH+). Using a computer-generated minimization procedure, patients were allocated to one of the two groups according to four preselected criteria: the number of embryos transferred, the cumulative score of transferred embryos, the age of the patient, and the use of ICSI. Results: Pregnancy and implantation rates in the AH+ and AH− groups were, respectively, 42.1 versus 38.1% and 17.9 versus 17.1%. Conclusions: From our data we conclude that assisted hatching through partial zona dissection prior to embryo transfer does not improve pregnancy and embryo implantation rates in unselected patients undergoing IVF or ICSI.

Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 micrograms/ml puromycin for 6-8 hr and further cultured for 12-15 hr.Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%).Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.

Our objective was to obtain parthenogenetic activation of unfertilized human oocytes by puromycin and to try to use this procedure for cytogenetic purposes.The setting was our IVF laboratory.Eighty-two spare oocytes from 34 IVF patients were used. In the first series of experiments 39 unfertilized oocytes were cultured in medium containing 100, 50, or 10 micrograms/ml puromycin for 6 to 24 hr. After the appearance of pronuclei they were transferred to plain medium, further cultured, and cytogenetically analyzed. In the second series of experiments 43 oocytes were cultured for 5 to 10 hr in 10 micrograms/ml puromycin, transferred to plain medium, and fixed for cytogenetic analysis 2 hr after nuclear envelope breakdown.Ninety-one percent of the oocytes in the first experiment showed the presence of one or more nuclei. From the pronucleate oocytes additionally cultured in puromycin-free medium, 46% developed further to the metaphase of the first mitotic division or the two-cell stage and 54% remained arrested at the pronuclear stage. In the second experiment 88% of the treated oocytes showed pronuclei or had cleaved, and after withdrawal from puromycin 96% of the pronucleate oocytes entered mitosis.Puromycin induces haploid as well as diploid parthenogenesis in aged human oocytes. A 5- to 10-hr treatment of oocytes with 10 micrograms/ml puromycin yields the highest percentage of activation, and almost all parthenogenetically activated oocytes enter or develop beyond the first cleavage mitosis. Analysis of mitotic metaphase chromosomes from parthenogenetically activated human oocytes may be a promising new approach to preimplantation cytogenetics.

The purpose of this study was to compare fertilization and aneuploidy rates after two stimulation protocols in an IVF program.This was a retrospective study.The study took place in the IVF laboratory of an Infertility Department.In 349 treatment cycles, clomiphene citrate (CC) and human menopausal gonadotropin (hMG) were used in one group (N = 233) and hMG after treatment with a gonadotropin-releasing hormone agonist (GnRHa) in two other groups (long protocol): goserelin (N = 73) and buserelin (N = 43). Cytogenetic analysis was performed on all unfertilized oocytes in both groups.Fertilization rates were significantly higher in the GnRHa/hMG group than in the CC/hMG group, but cleavage rates and embryo quality were not different. Of 736 oocytes prepared for cytogenetic analysis, 256 were karyotyped: 172 were found to be euploid and 84 aneuploid. More oocytes were aneuploid in the GnRHa/hMG group than in the CC/hMG group and this difference was statistically different after analysis of the data using a specially designed mathematical model.If no selection against chromosomally abnormal oocytes takes place at the time of fertilization, more abnormal oocytes are harvested with GnRHa/hMG protocols than with CC/hMG. If, on the other hand, there is a selection against oocytes with some chromosomal imbalance, there is no intrinsic effect of GnRH agonists on the chromosomal complement of the oocyte, and the real aneuploidy frequency in all oocytes, fertilized and unfertilized, is the same in the GnRHa/hMG and in the CC/hMG group.