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Study Question: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model?, Summary Answer: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring., What Is Known Already: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models., Study Design, Size, Duration: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx)., Participants/materials, Setting, Methods: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits., Main Results and the Role of Chance: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes., Large Scale Data: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318., Limitations, Reasons for Caution: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible., Wider Implications of the Findings: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes., Study Funding/competing Interest(s): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p = 0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p < 0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p = 0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Epidemiological studies suggest that babies born following in vitro fertilization (IVF) and fresh embryo transfer are of lower birthweight than babies born following frozen embryo transfer, although the mechanism responsible for this phenotype is not known. We developed a novel mouse model that isolates the independent effects of embryo freezing and the superovulated environment, which cannot be performed in humans. We transferred blastocysts that had been vitrified and warmed, mixed with with fresh blastocysts, into individual pseudopregnant recipients produced by either natural mating or mating following injection with equine chorionic gonadotropin and human chorionic gonadotropin and hCG (superovulation). We found that superovulation of the recipient dams led to significantly lower fetal weight at term while blastocyst vitrification had no significant effect on fetal weight (1.43 ± 0.24 g fresh-natural, 1.30 ± 0.28 g vitrified-natural vs. 1.09 ± 0.20 fresh-superovulated, 0.93 ± 0.23 g vitrified-superovulated, P < 0.0001). Doppler ultrasound revealed increased median umbilical artery resistance in the placentae of near-term dams exposed to superovulation compared to naturally mated dams (0.927 vs 0.904, P = 0.02). Additionally, placental microvascular density was lower in superovulated compared to naturally mated dams (1.24 × 10-3 vessel/micron vs 1.46 × 10-3 vessels/micron, P = 0.046). Gene expression profiling suggested alterations in fetal genes involved in glucorticoid regulation. These results suggest a potential mechanism for altered birthweight following superovulation in our model and may have implications for human IVF., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Assisted reproductive technologies (ART) have enabled millions of couples with compromised fertility to conceive children. Nevertheless, there is a growing concern regarding the safety of these procedures due to an increased incidence of imprinting disorders, premature birth, and low birth weight in ART-conceived offspring. An integral aspect of ART is the oxygen concentration used during in vitro development of mammalian embryos, which is typically either atmospheric (~20%) or reduced (5%). Both oxygen tension levels have been widely used, but 5% oxygen improves preimplantation development in several mammalian species, including that of humans. To determine whether a high oxygen tension increases the frequency of epigenetic abnormalities in mouse embryos subjected to ART, we measured DNA methylation and expression of several imprinted genes in both embryonic and placental tissues from concepti generated by in vitro fertilization (IVF) and exposed to 5% or 20% oxygen during culture. We found that placentae from IVF embryos exhibit an increased frequency of abnormal methylation and expression profiles of several imprinted genes, compared to embryonic tissues. Moreover, IVF-derived placentae exhibit a variety of epigenetic profiles at the assayed imprinted genes, suggesting that these epigenetic defects arise by a stochastic process. Although culturing embryos in both of the oxygen concentrations resulted in a significant increase of epigenetic defects in placental tissues compared to naturally conceived controls, we did not detect significant differences between embryos cultured in 5% and those cultured in 20% oxygen. Thus, further optimization of ART should be considered to minimize the occurrence of epigenetic errors in the placenta.

Culture systems that support development and maturation of oocytes in vitro with a high efficiency would have great impact not only on research addressed at underlying mechanisms of oocyte development but also on preservation of fertility. Recently, attention has turned to using culture systems that preserve follicle integrity, in contrast to existing systems that do not maintain follicle integrity, with the hope of improving oocyte development. We report that an alginate-based follicle culture system supports both follicular and oocyte growth in vitro, with little effect on the oocyte transcriptome. Nevertheless, oocytes obtained from these follicles exhibit an increased incidence of defects in spindle formation and chromosome alignment as well as pronounced abnormalities in cortical granule biogenesis. Developmental competence is also highly compromised, because few matured oocytes develop into 1-cell embryos with pronuclei. This situation contrasts with a high incidence of pronuclear formation following development using an existing in vitro culture system that does not preserve follicle integrity.

In the mammalian oocyte, the cAMP-dependent protein kinase (PKA) has critical functions in the maintenance of meiotic arrest and oocyte maturation. Because PKA is spatially regulated, its localization was examined in developing oocytes. Both regulatory subunits (RI and RII) and the catalytic subunit (C) of PKA were found in oocytes and metaphase II-arrested eggs. In the oocyte, RI and C were predominantly localized in the cortical region, while RII showed a punctate distribution within the cytoplasm. After maturation to metaphase II, RI remained in the cortex and was also localized to the meiotic spindle, while RII was found adjacent to the spindle. C was diffuse within the cytoplasm of the egg but was enriched in the cytoplasm surrounding the metaphase spindle, much like RII. The polarized localization and redistribution of RI, RII, and C suggested that PKA might be tethered by A-kinase anchor proteins (AKAPs), proteins that tether PKA close to its physiological substrates. An AKAP, AKAP140, was identified that was developmentally regulated and phosphorylated in oocytes and eggs. AKAP140 was shown to be a dual-specific AKAP, having the ability to bind both RI and RII. By compartmentalizing PKA, AKAP140 and/or other AKAPs could spatially regulate PKA activity during oocyte development.

The goal of fertilization is the union of one, and only one, sperm nucleus with the female pronucleus within the activated oocyte. For this to occur successfully, several events must transpire, including the incorporation of the entire spermatozoon into the oocyte, the completion of meiotic maturation with the extrusion of the second polar body, the metabolic activation of the previously quiescent oocyte, the decondensation of the sperm nucleus and the maternal chromosomes into the male and female pronuclei respectively, and the cytoplasmic migrations of the pronuclei, which bring them into apposition. Defects in any of these events are lethal to the zygote and might prove to be causes of infertility. In this study, the microtubules and DNA were imaged in inseminated human oocytes that had been discarded as unfertilized. The presence and number of incorporated sperm tails were also documented using a monoclonal antibody specific for the post-translationally modified acetylated-alpha-tubulin found in the tail, but not the oocyte, microtubules. An analysis of 211 oocytes from failed in-vitro fertilizations from 58 patient couples resulted in the determination of several previously undetectable phases at which fertilization arrests: (i) metaphase II arrest; (ii) arrest after the successful incorporation of the spermatozoon, (iii) arrest after the formation of the sperm aster; (iv) arrest during mitotic cell cycle progression; and (v) arrest during meiotic cell cycle progression. Data on polyspermy and arrested embryonic development are also presented. These results have implications for the diagnosis and treatment of female, as well as male, infertility.(ABSTRACT TRUNCATED AT 250 WORDS)

Although a number of different mechanisms have been suggested to account for the decline of fertility with age, the majority of studies agree that poor oocyte quality and reduced endometrial receptivity are the most important. In fact, the increased incidence of early pregnancy loss and chromosomal abnormalities of oocytes in older women, as well as the ability to reverse decreasing pregnancy rates by using oocyte donation, strongly support the evidence that oocyte ageing is the main factor responsible for decreasing fertility. Conversely, the lack of knowledge of the physiological variables that determine a successful nidation of a human embryo makes the analysis of uterine receptivity much more difficult. In order to evaluate the impact of the age of donors and recipients on pregnancy, implantation and abortion rates, we have retrospectively analysed 258 cycles from our programme of oocyte donation. Results were reviewed according to the following subclasses of age groups: < or = 30, 31-35 and 36-39 years for donors, and < or = 30, 31-35, 36-40, 41-45 and 46-53 years for recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

One of the limiting factors for the successful treatment of male sterility due to congenital absence of the vas deferens (CAVD) is the low (< 20%) and extremely unpredictable rate of in-vitro fertilization of their epididymal spermatozoa. The recent demonstration that CAVD is a mild form of cystic fibrosis (CF) disease, almost exclusively involving the genital tract, has prompted us to investigate the hypothesis of whether there could be an association between particular CF genotype mutations and the in-vitro performance of epididymal sperm. In this study 63 patients with surgically confirmed diagnosis of CAVD undergoing microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF) were evaluated. The genetic screening was carried out on DNA extracted from peripheral lymphocytes and amplified by the polymerase chain reaction. A total of 12 mutations in the cystic fibrosis transmembrane regulator gene (CFTR), representing approximately 90% of the total known CF mutations, were tested. The presence or absence of mutations, as well as the type of mutation found, was correlated with the IVF and pregnancy rates. Of the 63 patients examined, 40 (64%) were positive to the CF screening and 23 were negative. In three cases no spermatozoa were found because of rete testis blockage. The difference in fertilization rates between patients testing positive and negative was highly significant (P = 0.000003), 13 versus 23%, respectively. In order to pinpoint which mutation could account for the most deleterious effect on the IVF results, we analysed these by keeping the patients separated by CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated the cumulative pregnancy rate from one gamete intra-Fallopian transfer (GIFT) cycle plus subsequent cycles in which embryos cryopreserved at the time of the original GIFT cycle were transferred. All patients who had their first GIFT cycle in our centre between January, 1989 and March, 1991 were included. Ovarian stimulation was accomplished with leuprolide acetate (luteal phase protocol) and a combination of follicle stimulating hormone and human menopausal gonadotrophin. GIFT was performed with three to five oocytes. Excess oocytes were inseminated and good quality embryos were cryopreserved at the 2- to 4-cell stage with 1-2 propanediol. When the GIFT cycle did not result in a pregnancy, uterine transfer of cryopreserved embryos was carried out in subsequent unstimulated cycles. A total of 97 patients had GIFT and 46 pregnancies were achieved (47.4%). A total of 51 patients (52.5%) had embryos frozen; of these, 21 were from the non-pregnant group (41.1%) and 30 from the pregnant group (65.2%) (P < 0.05). Up to now, 22 of them have undergone a frozen-thawed embryo transfer cycle, and two of them achieved a pregnancy. Based on these data, patients having a GIFT could theoretically expect a cumulative pregnancy rate of 52.2%.

This report describes the first two pregnancies which occurred after cryopreservation, thawing and transfer of embryos generated using epididymal spermatozoa from men with congenital absence of the vas deferens. Of the 32 embryos that were thawed, 24 survived (75%) and were replaced in seven transfer cycles resulting in two (29%) pregnancies successfully delivered. The demonstration that excess embryos obtained with the use of epididymal spermatozoa can be safely frozen/thawed, and that their survival is not different from other embryos generated by in-vitro fertilization, represents a great advantage for the reproductive performance of men with congenital absence of the vas deferens undergoing microsurgical aspiration of epididymal spermatozoa.

We have analysed the different variables which affected the efficacy of egg donation in our assisted reproductive technology unit. Potential donors (n = 108) were divided into two groups. Group 1 comprised partial donor (PD) subjects who underwent assisted reproduction for the treatment of their infertility and who agreed altruistically to donate extra eggs not used during their own cycles (n = 31). Group 2 consisted of full donor (FD) subjects who donated all the eggs recovered during their ovarian stimulation cycle (n = 77) and included sister to sister donation, volunteers and patients undergoing diagnostic or sterilization laparoscopies. The entire donor population was divided into two sub-populations by age, those under 30 and 31-39 years old, and correlated with the pregnancy rate in their recipients. From this analysis, a higher pregnancy rate was observed when the oocytes were provided by the younger group of donors (54 versus 36%). We did not find a significant difference in the recipients' pregnancy rate, according to the previous reproductive performance of their donors. Even though FD and PD yield similar pregnancy rates during the fresh cycles, the availability of cryopreserved embryos in the former group enhances the cumulative pregnancy rate.

Conventional methods of semen preparation for IVF, such as swim-up sedimentation, regular Percoll gradients, are not very effective in the treatment of severe oligoasthenozoospermia (total motile count less than 5 X 10(6)). For these cases, a new method of sperm preparation consisting of a reduced volume of a discontinuous Percoll gradient (mini-Percoll) has been utilized. The results obtained were analysed by comparing mini-Percoll versus resuspension in two groups of patients with severe male factor infertility. In the mini-Percoll group, a statistically significant improvement (P less than 0.001) was obtained in the post-treatment seminal parameters of motility, progression and proportion of normal forms. In the resuspension group (n = 12), only eight of 108 oocytes inseminated were fertilized (7%), while in the mini-Percoll group (n = 29), 124 out of 336 oocytes were fertilized (40%) (P less than 0.001). Furthermore, in the mini-Percoll group, a higher pregnancy rate (25 versus 7%) and low abortion rate (22 versus 33%) were observed. Although more studies are necessary to confirm these preliminary data, mini-Percoll seems to be a valid laboratory tool for semen samples of extremely poor quality.

Selective cannulation of the Fallopian tubes via the utero-tubal junction, without analgesia, anaesthesia or surgery, has been carried out to replace three embryos in a patient. Cannulating the Fallopian tubes did not appear materially to affect the transport of embryos or the receptivity of the endometrium for implantation.

We have combined intrauterine insemination (IUI) and controlled ovarian hyperstimulation (COH), for the treatment of infertility due to different aetiologies, prior to performing GIFT. To date, we have treated 186 patients over a total of 489 cycles. The mean age of the patients was 34.1 +/- 4 years and the mean duration of infertility was 4.8 +/- 2.8 years. Follicular development was induced with human menopausal gonatrophin (HMG). Patients were monitored using serum oestradiol determinations and ovarian ultrasound. Two intrauterine inseminations were performed 12 and 36 h after HCG injection. Semen samples were prepared utilizing one of two techniques, swim-up or Percoll gradient. A total of 33 pregnancies have occurred, the gross pregnancy rate being 17.7% per patient and 6.7% per cycle. The cumulative pregnancy rate was 30%. Thirty-one pregnancies (94%) occurred within the first four cycles of treatment. During the same period of time, the pregnancy rate per cycle in patients treated with gamete intra-Fallopian transfer (GIFT) was 32.9%. Our data suggest that IUI combined with COH can result in pregnancy in a significant proportion of patients, but that the efficiency per cycle of the technique is significantly lower than GIFT.

To date the main limitation of in-vitro fertilization-embryo transfer (IVF-ET) programmes is that transcervical transfer of embryos results in a rate of low implantation. On the other hand, the technique of gamete intra-Fallopian transfer (GIFT) does not contribute to information on oocyte fertilization rates, and the time of oocyte exposure to sperm may be limited. The development of ultrasonically guided follicular aspiration will allow transfer of embryos generated in vitro to the Fallopian tubes performing only one surgical procedure in the process. We have performed 25 intra-tubal embryo transfers in the cynomolgus monkey (Macaca fascicularis). Ovarian stimulation, follicular aspiration and IVF procedures have been reported previously by Balmaceda. ETs were performed via laparotomy. Embryos at the 2-, 4- or 9-cell stage were loaded into a tom-cat catheter in 5 microliter of culture medium and delivered to the mid-ampullary portion of the tube. Seven ETs performed during stimulated cycles resulted in one pregnancy, and 18 ETs performed in synchronized recipients resulted in six pregnancies. Ten ETs were performed 0-24 h, eight performed 24-48 h and seven performed 72-110 h after follicular aspiration or ovulation, and resulted in 4, 3 and 0 pregnancies respectively. Our results demonstrate that intra-tubal embryo transfer can result in normal intra-uterine pregnancies and suggest that both ovarian stimulation and cycle synchronization affect the probability of embryo implantation.

The outcome of a series of 19 patients with premature ovarian failure (POF) undergoing gamete intra-Fallopian transfer (GIFT), utilizing donated oocytes, is described. The steroid replacement protocol consisted of the administration of increasing dosages of 17 beta-oestradiol (E2) and progesterone (P4). Hormonal replacement was maintained until day 100 of gestation. All patients underwent an evaluation cycle in which serum levels of E2 and P4 were monitored and an endometrial biopsy was performed either 7 or 11 days after initiation of progesterone administration. All cases of GIFT were performed between days 12 and 15 of the induced menstrual cycle. Of the 19 patients treated, 11 became pregnant, giving a clinical pregnancy rate of 58% (visualization of gestational sac by ultrasound). Two patients aborted between the 4th and 5th weeks of gestation. No ectopic pregnancies occurred. Three of the seven deliveries involved multiple births. Details of the circulating hormone levels and endometrial response are discussed.

The outcome of a series of 115 patients undergoing GIFT by minilaparotomy is described, their duration of infertility ranging between 3 and 19 years. Ovarian stimulation was carried out using clomiphene from day 3 and HMG from day 6, and HCG was used to induce follicular maturation. A maximum of four oocytes, two per oviduct with 100,000 spermatozoa, were replaced. Thirty-six pregnancies were established. The highest incidence of pregnancy was obtained in cases of unexplained infertility (42%) and endometriosis (28%), and the lowest with male factors (13%). The most mature oocytes gave the best results. There were eleven miscarriages (30.5%) and two ectopic pregnancies (5.5%). Eleven of the 23 deliveries involved multiple births.