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1.
Hum Reprod Open ; 2022(3): hoac027, 2022.
Article in English | MEDLINE | ID: mdl-35795849

ABSTRACT

STUDY QUESTION: Does intraovarian injection of platelet-rich plasma (PRP) change ovarian function in patients with extremely low functional ovarian reserve (LFOR) who, otherwise, would likely only have a chance of pregnancy through third-party oocyte donation? SUMMARY ANSWER: No clinically significant effects of PRP treatment on ovarian function were observed over 1 year of follow-up. WHAT IS KNOWN ALREADY: Several investigators have reported improved responses to ovulation induction after treatment with PRP. However, previous published reports have involved, at most, only small case series. Whether PRP actually improves ovarian performance is, therefore, still unknown. PRP is nevertheless widely offered as an 'established' fertility treatment, often under the term 'ovarian rejuvenation'. STUDY DESIGN SIZE DURATION: We are reporting a prospective cohort study of 80 consecutive patients at ages 28-54 with LFOR, defined by anti-Müllerian hormone <1.1 ng/ml, FSH >12 mIU/ml or at least one prior IVF cycle with ≤3 oocytes within 1 year. The women were followed for 1 year after an intraovarian PRP procedure. PARTICIPANTS/MATERIALS SETTING METHODS: PRP (1.5 ml) was injected into the cortex of ovaries with an average of 12 injections per ovary. Study participants were followed every 3 days for 2 weeks after PRP treatment with estradiol and FSH measurements and vaginal ultrasound to observe follicle growth and thereafter followed weekly. Beginning 1 month after their PRP treatment, participants underwent one or more cycles of ovarian stimulation for IVF. Outcome measures were endocrine response, and numbers of oocytes and embryos produced in response to a maximal gonadotropin stimulation before and after PRP treatment. MAIN RESULTS AND THE ROLE OF CHANCE: In this study, women failed to demonstrate statistically significant outcome benefits from intraovarian PRP. However, two 40-year-old very poor-prognosis patients, with prior failed IVF cycles that never reached embryo transfer at other centers, achieved pregnancy, resulting in an ongoing pregnancy rate of 4.7% among patients who, following PRP, produced at least one oocyte (n = 42). LIMITATIONS REASONS FOR CAUTION: As an observational study of patients who performed poorly in past ovarian stimulation cycles, the improvement may be accounted for by regression to the mean. Similar considerations may also explain the occurrence of the two pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrates that, even in extremely poor prognosis patients due to LFOR, sporadic pregnancies are possible. The study, however, does not allow for the conclusion that those pregnancies were the consequence of PRP treatments. A case series, indeed, does not allow for such conclusions, even if results are more suggestive than here. This registered study, therefore, must be viewed as a preliminary report, with further data expected from this study but also from two other prospectively randomized ongoing registered studies with more controlled patient selection. STUDY FUNDING/COMPETING INTERESTS: This work was supported by intramural funds from The Center for Human Reproduction and the not-for-profit research Foundation for Reproductive Medicine, both in New York, NY, USA. N.G. and D.H.B. are listed as co-inventors on several US patents. Some of these patents relate to pre-supplementation of hypo-androgenic infertile women with androgens, such as dehydroepiandrosterone and testosterone and, therefore, at least peripherally relate to the subject of this manuscript. They, as well as D.F.A., have also received research support, travel funds and speaker honoraria from several pharmaceutical and medical device companies, though none related to the here presented subject and manuscript. N.G. is a shareholder in Fertility Nutraceuticals and he and D.H.B. receive royalty payments from Fertility Nutraceuticals LLC. E.M. has no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NCT04275700.

3.
Hum Reprod ; 37(6): 1194-1206, 2022 05 30.
Article in English | MEDLINE | ID: mdl-35413106

ABSTRACT

STUDY QUESTION: What are the outcomes for patients who choose to move embryos diagnosed as abnormal by preimplantation genetic testing for aneuploidy (PGT-A) to a new institution for transfer after the diagnosing institution refused to transfer them? SUMMARY ANSWER: Many patients seek to have selected embryos with PGT-A abnormal trophectoderm biopsies transferred recognizing that these embryos can still offer a chance of pregnancy and live birth. WHAT IS KNOWN ALREADY: : PGT-A is a widely practiced method of selecting embryos for transfer based on biopsy of a few cells. Many clinical practices refuse to transfer PGT-A abnormal embryos even when there are no other 'normal' embryos available. STUDY DESIGN, SIZE, DURATION: This is a prospective cohort of 69 couples who, since 2014, moved a total of 444 PGT-A abnormal embryos previously refused transfer at their parent institutions to our practice. Among these, 50 patients have, thus far, undergone 57 transfer cycles of 141 embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos diagnosed at other institutions by PGT-A as abnormal (mostly using next generation sequencing) were moved to our academically affiliated private fertility and research center in New York City. Female age at retrieval was 41.35 ± 3.98 years, 74% were Caucasian, 12% Asian and 10% were of African descent. All embryos identified as PGT-A abnormal among prospectively identified couples were recorded in our center's registry. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 144 embryos transferred 102 (72.3%) had only 1 or 2 chromosomal abnormalities, 30 (21.3%) had 3 or more and 9 (6.4%) were 'undiagnosed' because of degraded DNA, yet still had been refused transfer. Transfer of PGT-A abnormal embryos resulted in 8 live births, 11 miscarriages and no voluntary terminations. One child was born with a segmental duplication and required repair of coarctation of the aorta as a newborn. Many couples with only PGT-A abnormal embryos are willing to have their PGT-A abnormal embryos transferred and such transfers can result in the establishment of ongoing euploid pregnancies and live births. LIMITATIONS, REASONS FOR CAUTION: Findings in this case series represent couples who chose to have their embryos transferred after having been refused transfer elsewhere and may not be representative of the wider population of couples undergoing IVF with PGT-A in general. Not all abnormal phenotypes present in the immediate postnatal period so it will be important to continue to follow the development of these children. WIDER IMPLICATIONS OF THE FINDINGS: PGT-A can result in a clinics refusal to transfer embryos with abnormal PGT-A biopsies, even those with mosaic findings, consequently large numbers of infertile women are prematurely advised that their only chance of motherhood is through third-party egg-donation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funds from the Center for Human Reproduction and the not-for-profit research Foundation for Reproductive Medicine, both in New York, NY, USA. N.G. and D.H.B. are listed as co-inventors on several U.S. patents. One of these patents (US Patent# 7,615,544) relates to pre-supplementation of hypo-androgenic infertile women with androgens, such as DHEA and testosterone and, therefore, at least peripherally related to the subject of this manuscript. N.G. and D.F.A. also received travel funds and speaker honoraria from several pharmaceutical and medical device companies, though none related to the here presented subject and manuscript. N.G. is a shareholder in Fertility Nutraceuticals and he and D.H.B. receive royalty payments from Fertility Nutraceuticals LLC. TRIAL REGISTRATION NUMBER: N/A.


Subject(s)
Infertility, Female , Preimplantation Diagnosis , Aneuploidy , Biopsy , Cohort Studies , Female , Fertilization in Vitro , Genetic Testing/methods , Humans , Male , Pregnancy , Preimplantation Diagnosis/methods , Prospective Studies
4.
Hum Reprod Open ; 2020(4): hoaa049, 2020.
Article in English | MEDLINE | ID: mdl-33381657

ABSTRACT

STUDY QUESTION: Does the ovarian sensitivity index (OSI) predict embryo quality, pregnancy and live birth in patients undergoing FSH/hMG stimulation for IVF? SUMMARY ANSWER: The OSI is predictive of pregnancy and live birth in older women with a more unfavorable prognosis undergoing FSH/hMG stimulation for IVF. WHAT IS KNOWN ALREADY: The OSI was previously reported to reflect gonadotrophin requirements among high, normal and poor responders and to predict pregnancy potential in younger patients undergoing ovarian stimulation with FSH. STUDY DESIGN SIZE DURATION: A retrospective cohort study that included 1282 women undergoing IVF with FSH/hMG stimulation was carried out between January 2010 and December 2016. PARTICIPANTS/MATERIALS SETTING METHODS: We evaluated 1282 women who underwent fertility treatment with FSH/hMG stimulation and oocyte retrieval at an academically affiliated private fertility center. OSI was calculated as (oocytes ×1000)/total gonadotrophin dose and grouped into two classes based on a receiver operating characteristic (ROC) curve analysis of a randomly selected development sample comprising one-third of the cycles. The remaining cycles comprised the validation group. ROC curves were also used to compare the predictive value of OSI to that of baseline FSH and anti-Müllerian hormone (AMH). Logistic regression models evaluated the effect of high (OSI >0.83) and low (OSI ≤0.83) on clinical pregnancy and live birth in the validation group. Models were adjusted for female age, baseline FSH, AMH and oocyte yield and gonadotrophin dose. MAIN RESULTS AND THE ROLE OF CHANCE: Women presented with a mean ±SD age of 38.6 ± 5.4 years and showed median AMH levels of 0.65 (95% CI 0.61-0.74) ng/ml. They received 5145 ± 2477 IU of gonadotrophins and produced a median 5.2 (95% CI 5.0-5.5) oocytes. Pregnancy and live birth rates per oocyte retrieval for all women were 20.6% and 15.8%, respectively. Patients with higher OSI (less gonadotrophin required per oocyte retrieved) produced significantly more high-quality embryos than patients with low OSI (3.5 (95% CI 3.2-3.8) versus 0.6 (95% CI 0.5-0.7) (P = 0.0001)) and demonstrated higher pregnancy (23.2% versus 9.7%) and live birth rates (8.8% versus 5.3%) than their counterparts (P = 0.0001 and P = 0.0001, respectively). After adjustments for age, baseline AMH and FSH, total gonadotrophin dosage and oocyte yield, an OSI >0.83 was associated with greater odds of pregnancy (odds ratio 2.12, 95% CI 1.30-3.45, P < 0.003) and live birth (odds ratio 1.91, 95% CI 1.07-3.41, P < 0.028). LIMITATIONS REASONS FOR CAUTION: The results may not be applicable to women with excellent pregnancy potential or FSH-only stimulation. WIDER IMPLICATIONS OF THE FINDINGS: The predictive capacity of OSI for embryo quality, pregnancy and live birth, which is independent of AMH or FSH, may help in counseling patients about their pregnancy potential and live birth chances. STUDY FUNDING/COMPETING INTERESTS: Intramural funding from the Center for Human Reproduction and the Foundation for Reproductive Medicine. A.W., V.A.K., D.F.A., D.H.B. and N.G. have received research grant support, travel funds and speaker honoraria from various pharmaceutical and medical device companies: none, however, related to the topic presented here. D.H.B. and N.G. are listed as inventors on already awarded and still pending US patents, claiming beneficial effects on diminished ovarian reserve and embryo ploidy from dehydroepiandrosterone supplementation. TRIAL REGISTRATION NUMBER: N/A.

5.
J Assist Reprod Genet ; 37(9): 2259-2271, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32613414

ABSTRACT

PURPOSE: The age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep. METHODS: The expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70-90 µm (S), 90-110 µm (M), or 110-130 µm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years). RESULTS: Specific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences. CONCLUSION: Our study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.


Subject(s)
Embryonic Development/genetics , Multiprotein Complexes/genetics , Oogenesis/genetics , Ovarian Follicle/growth & development , Animals , DNA-Binding Proteins , Female , Gene Expression Regulation, Developmental/genetics , Maternal Age , Oocytes/growth & development , Oocytes/metabolism , Ovarian Follicle/metabolism , RNA, Messenger/genetics , Sheep/genetics , Sheep/growth & development
6.
Reprod Biol Endocrinol ; 18(1): 57, 2020 May 29.
Article in English | MEDLINE | ID: mdl-32471441

ABSTRACT

BACKGROUND: A recently published Position Statement (PS) by the Preimplantation Genetics Diagnosis International Society (PGDIS) regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF) contained inaccuracies and misrepresentations. Because opinions issued by the PGDIS have since 2016 determined worldwide IVF practice, corrections appear of importance. METHODS: The International Do No Harm Group in IVF (IDNHG-IVF) is a spontaneously coalesced body of international investigators, concerned with increasing utilization of add-ons to IVF. It is responsible for the presented consensus statement, which as a final document was reached after review of the pertinent literature and again revised after the recent publication of the STAR trial and related commentaries. RESULTS: In contrast to the PGDIA-PS, we recommend restrictions to the increasing, and by IVF centers now often even mandated, utilization of PGT-A in IVF cycles. While PGT-A has been proposed as a tool for achieving enhanced singleton livebirth outcomes through embryo selection, continued false-positive rates and increasing evidence for embryonic self-correction downstream from the testing stage, has led IDNHG-IVF to conclude that currently available data are insufficient to impose overreaching recommendations for PGT-A utilization. DISCUSSION: Here presented consensus offers an alternative to the 2019 PGDIS position statement regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF). Mindful of what appears to offer best outcomes for patients, and in full consideration of patient autonomy, here presented opinion is based on best available evidence, with the goal of improving safety and efficacy of IVF and minimizing wastage of embryos with potential for healthy births. CONCLUSIONS: As the PGDIS never suggested restrictions on clinical utilization of PGT-A in IVF, here presented rebuttal represents an act of self-regulation by parts of the IVF community in attempts to control increasing utilization of different unproven recent add-ons to IVF.


Subject(s)
Aneuploidy , Embryo Transfer/standards , Fertilization in Vitro , Mosaicism , Preimplantation Diagnosis/standards , Blastocyst , False Positive Reactions , Female , Humans , Pregnancy
7.
PLoS One ; 14(10): e0222390, 2019.
Article in English | MEDLINE | ID: mdl-31647816

ABSTRACT

The size of oocytes was previously reported to be smaller in obese women with polycystic ovary syndrome (PCOS). In the present prospective cohort study, we sought to determine whether oocyte size and morphology are associated with patient characteristics in non-PCOS women. Oocyte and oolemmal diameter were measured, enlarged perivitelline space (PVS) and ooplasmic granulation were assessed in 308 MII oocytes from 77 IVF/ICSI couples. Statistical analysis was undertaken using SAS version 9.4 (SAS institute Inc., USA). Continuous values are presented as mean ± SD and compared using a two-sample t-test or Mann-Whitney U test as appropriate. Categorical parameters are presented as proportions and compared using a Fisher exact test. Logistic and linear regression models were used to control for the effect of age for categorical and continuous variables respectively. P-value < 0.05 was considered statistically significant. Patients presented with a mean age of 40.3±5.0 years, had a BMI of 25.1±6.1 kg/m2, median AMH levels of 0.6 ng/ml and produced a median of 4 oocytes. Mean total oocyte diameter was 163.2±7.4 µm (range 145.8-182.1 µm), while oolemmal diameter was 109.4±4.1 µm (range 98.5-122.3 µm). After adjusting for age and ovarian reserve increasing BMI was associated with decreased total oocyte diameter (p<0.05). Total oocyte diameter was also inversely associated with AMH levels (p = 0.03) and oocyte yield (p = 0.04). In contrast to total oocyte diameter, oolemmal diameter was not related to patient characteristics. Younger women and those with large oocyte yields demonstrated fewer oocytes with ooplasmic granulation (p<0.05 and p = 0.01). After adjustments for age, ooplasmic granulation was also less frequently observed in oocytes from women with higher AMH (p = 0.03) and increasing BMI (p<0.01). Fertilization was more likely in oocytes with larger oolemmal diameter (p = 0.008). Embryos from oocytes with larger total and ooplasmic diameters were more likely to be transferred or frozen (p = 0.004 and p = 0.01). In non-PCOS infertile women, BMI and ovarian function relate to total oocyte diameter. These results expand on previously observed associations between oocyte size and BMI in women with PCOS. They indicate the importance of detailed oocyte assessments, which may aid the currently used criteria for embryo selection and help to better understand how oocyte status is associated with later embryo development.


Subject(s)
Cell Size , Infertility, Female/therapy , Oocytes/growth & development , Ovarian Reserve/physiology , Adult , Body Mass Index , Embryonic Development/physiology , Female , Fertilization in Vitro , Humans , Infertility, Female/physiopathology , Male , Middle Aged , Oocyte Retrieval/methods , Oocytes/pathology , Ovulation Induction , Polycystic Ovary Syndrome/pathology , Pregnancy , Pregnancy Rate , Sperm Injections, Intracytoplasmic
8.
J Assist Reprod Genet ; 35(5): 735-751, 2018 May.
Article in English | MEDLINE | ID: mdl-29497954

ABSTRACT

An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.


Subject(s)
Cumulus Cells , Follicular Fluid/physiology , Oocytes/physiology , Animals , Cumulus Cells/cytology , Cumulus Cells/physiology , Diabetes Mellitus/pathology , Endometriosis/pathology , Female , Follicular Fluid/cytology , Humans , Infertility, Female/etiology , Infertility, Female/pathology , Obesity/complications , Obesity/pathology , Oocytes/cytology , Pelvic Infection/complications , Pelvic Infection/pathology , Polycystic Ovary Syndrome , Pregnancy
9.
Mol Hum Reprod ; 24(3): 135-142, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29390119

ABSTRACT

STUDY QUESTION: Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? SUMMARY ANSWER: Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. WHAT IS KNOWN ALREADY: Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. STUDY DESIGN, SIZE, DURATION: Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). PARTICIPANTS/MATERIALS, SETTING, METHODS: Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 µm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 µm diameter were selected for IVM in SAGE medium (Step 4) then fixed for analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 µm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end of Step 3, 32 complexes contained oocytes >100 µm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. LIMITATIONS, REASONS FOR CAUTION: This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimization with morphological evaluation and fertilization potential of IVG oocytes is required to determine whether they are normal. WIDER IMPLICATIONS OF THE FINDINGS: The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilization would benefit fertility preservation practice. STUDY FUNDING/COMPETING INTEREST(S): Funded by MRC Grants (G0901839 and MR/L00299X/1). No competing interests.


Subject(s)
Cell Culture Techniques/methods , Oocytes/cytology , Ovarian Follicle/cytology , Ovary/cytology , Ovary/metabolism , Adult , Female , Humans , Meiosis/genetics , Meiosis/physiology , Oogenesis/genetics , Oogenesis/physiology
10.
Nanotechnology ; 28(45): 455602, 2017 11 10.
Article in English | MEDLINE | ID: mdl-29039364

ABSTRACT

Pt nanoparticles in a Al2O3 dielectric matrix thin films are elaborated by means of atomic layer deposition. These nanostructured thin films are integrated in vertical and planar test structures in order to assess both their in-plane and out-of-plane electrical properties. A shadow edge evaporation process is used to develop planar devices with electrode separation distances in the range of 30 nm. Both vertical and planar test structures show a Poole-Frenkel conduction mechanism. Low trap energy levels (<0.1 eV) are identified for the two test structures which indicates that the Pt islands themselves are not acting as traps in the PF mechanism. Furthermore, a more than three order of magnitude current density difference is observed between the two geometries. This electrical anisotropy is attributed to a large electron mobility difference in the in-plane and out-of-plane directions which can be related to different trap distributions in both directions.

11.
J Assist Reprod Genet ; 33(11): 1431-1438, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27525657

ABSTRACT

The subcortical maternal complex (SCMC) is a multiprotein complex uniquely expressed in mammalian oocytes and early embryos, essential for zygote progression beyond the first embryonic cell divisions. Similiar to other factors encoded by maternal effect genes, the physiological role of SCMC remains unclear, although recent evidence has provided important molecular insights into different possible functions. Its potential involvement in human fertility is attracting increasing attention; however, the complete story is far from being told. The present mini review provides an overview of recent findings related to the SCMC and discusses its potential physiological role/s with the aim of inspiring new directions for future research.


Subject(s)
Embryonic Development/genetics , Fertility/genetics , Multiprotein Complexes/genetics , Oocytes/metabolism , Amino Acid Sequence/genetics , Blastocyst/metabolism , Female , Gene Expression Regulation, Developmental , Humans , Oocytes/growth & development , Zygote/growth & development , Zygote/metabolism
12.
Hum Reprod ; 29(1): 97-106, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24135076

ABSTRACT

STUDY QUESTION: Do the ovarian follicles of children and adolescents differ in their morphology and in vitro growth potential from those of adults? SUMMARY ANSWER: Pre-pubertal ovaries contained a high proportion of morphologically abnormal non-growing follicles, and follicles showed reduced capacity for in vitro growth. WHAT IS KNOWN ALREADY: The pre-pubertal ovary is known to contain follicles at the early growing stages. How this changes over childhood and through puberty is unknown, and there are no previous data on the in vitro growth potential of follicles from pre-pubertal and pubertal girls. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five pre-pubertal and seven pubertal girls and 19 adult women were analysed histologically, cultured in vitro for 6 days, with growing follicles then isolated and cultured for a further 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were obtained from girls undergoing ovarian tissue cryopreservation for fertility preservation, and compared with biopsies from adult women. Follicle stage and morphology were classified. After 6 days in culture, follicle growth initiation was assessed. The growth of isolated secondary follicles was assessed over a further 6 days, including analysis of oocyte growth. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-pubertal ovaries contained a high proportion of abnormal non-growing follicles (19.4 versus 4.85% in pubertal ovaries; 4004 follicles analysed; P = 0.02) characterized by indistinct germinal vesicle membrane and absent nucleolus. Follicles with this abnormal morphology were not seen in the adult ovary. During 6 days culture, follicle growth initiation was observed at all ages; in pre-pubertal samples there was very little development to secondary stages, while pubertal samples showed similar growth activation to that seen in adult tissue (pubertal group: P = 0.02 versus pre-pubertal, ns versus adult). Isolated secondary follicles were cultured for a further 6 days. Those from pre-pubertal ovary showed limited growth (P < 0.05 versus both pubertal and adult follicles) and no change in oocyte diameter over that period. Follicles from pubertal ovaries showed increased growth; this was still reduced compared with follicles from adult women (P < 0.05) but oocyte growth was proportionate to follicle size. LIMITATIONS, REASONS FOR CAUTION: These data derive from only a small number of ovarian biopsies, although large numbers of follicles were analysed. It is unclear whether the differences between groups are related to puberty, or just age. WIDER IMPLICATIONS OF THE FINDINGS: These findings show that follicles from girls of all ages can be induced to grow in vitro, which has important implications for some patients who are at high risk of malignant contamination of their ovarian tissue. The reduced growth of isolated follicles indicates that there are true intrafollicular differences in addition to potential differences in their local environment, and that there are maturational processes occurring in the ovary through childhood and adolescence, which involve the loss of abnormal follicles, and increasing follicle developmental competence. STUDY FUNDING/COMPETING INTEREST(S): Funded by MRC grants G0901839 and G1100357. No competing interests.


Subject(s)
Ovarian Follicle/physiology , Sexual Maturation/physiology , Adolescent , Adult , Biopsy , Child , Child, Preschool , Cryopreservation , Female , Fertility Preservation , Humans , Oocytes/growth & development , Oocytes/pathology , Ovarian Follicle/growth & development , Ovary/pathology , Puberty , Tissue Culture Techniques
14.
Mol Hum Reprod ; 16(9): 644-53, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20203128

ABSTRACT

The aim of this study was to determine the individual and combined effect of activin and follicle stimulating hormone (FSH) on somatic and germ cell development in cultured pre-antral follicles. Pre-antral bovine follicles (mean diameter 157 +/- 3, range 132-199 microm) were cultured for 8 days in serum-free medium in the presence of either 100 ng/ml of recombinant human activin A (rhAct A), 100 ng/ml rhAct A combined with a high (100 ng/ml) or low (50 ng/ml) concentration of recombinant FSH (rFSH) or 50 ng/ml rFSH alone. Intrafollicular connexin 43 expression and actin-based cell adhesion were assessed on Day 2 and 4 of culture. Steroidogenesis was evaluated after Day 4 and 8. Follicles exposed to 100 ng/ml activin maintained expression of connexin 43 at the follicular periphery. In the presence of activin, with or without 100 ng/ml or 50 ng/ml FSH, follicles were steroidogenic undergoing significant growth (P < 0.01), granulosa cell proliferation (P < 0.01) and antral cavity formation (P < 0.05) compared with cultured controls. Maximum oocyte growth occurred in the presence of 100 ng/ml activin alone with a significant percentage of these oocytes maintaining normal morphology over controls (P < 0.05). These results are consistent with a role for activin in maintaining oocyte granulosa cell interactions due to increased peripheral granulosa cell adhesion to the basement membrane and retention of adhesion at the surface of the zona pellucida. Thus, the polarized expression of cell contact interactions promoted by activin supports ongoing folliculogenesis.


Subject(s)
Activins/metabolism , Inhibin-beta Subunits/metabolism , Oocytes/metabolism , Oogenesis , Ovarian Follicle/metabolism , Actins/metabolism , Animals , Basement Membrane/metabolism , Cattle , Cell Adhesion , Cell Communication , Cell Polarity , Cell Proliferation , Cell-Matrix Junctions/metabolism , Cells, Cultured , Connexin 43/metabolism , Culture Media, Conditioned/metabolism , Estradiol/metabolism , Female , Follicle Stimulating Hormone/metabolism , Granulosa Cells/metabolism , Humans , Ki-67 Antigen/metabolism , Ovarian Follicle/cytology , Recombinant Proteins/metabolism , Time Factors
15.
Reprod Biomed Online ; 19 Suppl 3: 29-34, 2009.
Article in English | MEDLINE | ID: mdl-20034421

ABSTRACT

The metaphase II (MII) spindle of the human oocyte may be damaged by cryopreservation. High performance confocal microscopy was used to assess meiotic spindle and chromosome organization in oocytes after vitrification by the cryoleaf system. Three hours after retrieval, donor mature oocytes were fixed or vitrified. Vitrification was performed by equilibration in 7.5% ethylene glycol (EG) and 7.5% dimethylsulphoxide (DMSO), transfer to 15% EG, 15% DMSO and 0.5 mol/l sucrose, and loading onto cryoleaf strips. Tubulin staining was found in all survived vitrified-warmed oocytes, the majority (62.8%) of which displayed a bipolar spindle. A normal bipolar spindle configuration and equatorial chromosome alignment was observed only in a part of vitrified-warmed oocytes (32.6%). This frequency was significantly lower in comparison to fresh oocytes (59.1%). In another fraction of vitrified-warmed oocytes (30.2%), spindle bipolarity was associated to one or more non-aligned scattered chromosomes that often appeared tenuously associated with the lateral microtubules of the spindle. Furthermore, in cryopreserved oocytes with a bipolar spindle, a significantly increased pole-to-pole distance (14.9 +/- 2.3 microm) was found in comparison to the fresh control (12.4 +/- 2.6 microm) (P = 0.001). Therefore, under the conditions tested, vitrified-warmed oocytes maintain a MII spindle with a bipolar organization. However, chromosome alignment appears to be partly compromised.


Subject(s)
Chromosome Aberrations/drug effects , Cryopreservation , Cryoprotective Agents/adverse effects , Metaphase/drug effects , Oocytes/drug effects , Spindle Apparatus/drug effects , Adult , Chi-Square Distribution , Embryo Culture Techniques , Female , Fluorescent Antibody Technique , Humans , Image Processing, Computer-Assisted , Metaphase/genetics , Microscopy, Confocal , Oocytes/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Tubulin/metabolism
16.
Hum Reprod ; 24(9): 2114-23, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19465461

ABSTRACT

BACKGROUND: The demand for cryopreservation of human oocytes is increasing in assisted reproduction clinics and yet remains an experimental procedure. Surprisingly, little is known about the effects of cryopreservation on spindle-chromosome interactions and the recovery of meiotic spindle functionality. The goal of these studies was to evaluate the process of meiotic spindle reassembly and chromosome alignment in cryopreserved human metaphase II oocytes. METHODS: Unfrozen control oocytes were compared with frozen oocytes fixed at 0, 1, 2 and 3 h after thawing. Oocytes were analysed by confocal microscopy and subjected to 3-dimensional image analysis to evaluate spindle integrity. RESULTS: Freezing resulted in a loss of spindle bipolarity and chromosome alignment. One hour following thawing, most oocytes recovered spindle bipolarity and equatorial chromosomal alignment. However, between 2 and 3 h, a progressive loss of chromosome alignment was observed. Further analysis revealed a positive correlation between spindle length and number of displaced chromosomes following freezing. This time-dependent redistribution of chromosomes involved outward displacement from the equatorial plate and retention at the surface of the meiotic spindle. CONCLUSIONS: Spindle disassembly incurred by cryopreservation is rapidly reversed and is coordinated with chromosome alignment within 1 h but is not sustained at later times.


Subject(s)
Cryopreservation/methods , Meiosis/physiology , Oocytes/physiology , Acetylation , Adult , Chromosomes, Human/physiology , Chromosomes, Human/ultrastructure , Female , Humans , Male , Microscopy, Confocal , Tubulin/metabolism
17.
J Cell Physiol ; 216(2): 355-65, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18452183

ABSTRACT

Oogenesis serves a singular role in the reproductive success of plants and animals. Of their remarkable differentiation pathway what stands out is the ability of oocytes to transform from a single cell into the totipotent lineages that seed the early embryo. As our understanding that commonalities between diverse organisms at the genetic, cellular and molecular levels are conserved to achieve successful reproduction, the notion that embryogenesis presupposes oogenesis has entered the day-to-day parlance of regenerative medicine and stem cell biology. With emphasis on the mammalian oocyte, this review will cover (1) current concepts regarding the birth, survival and growth of oocytes that depends on complex patterns of cell communication between germ line and soma, (2) the notion of "maternal inheritance" from a genetic and epigenetic perspective, and (3) the relative value of model systems with reference to current clinical and biotechnology applications.


Subject(s)
Embryo, Mammalian , Oocytes , Oogenesis , Animals , Cell Communication/physiology , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/physiology , Embryonic Development/physiology , Feedback, Physiological , Female , Humans , Oocytes/cytology , Oocytes/physiology , Oogenesis/physiology , Ovarian Follicle/cytology , Ovarian Follicle/physiology , Ovulation , Pregnancy
18.
Hum Reprod ; 22(10): 2776-83, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17675355

ABSTRACT

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.


Subject(s)
Cell Membrane Permeability , Cryopreservation/methods , Ethylene Glycol/metabolism , Oocytes/physiology , Cell Survival , Female , Humans , Male , Oocytes/drug effects , Oocytes/ultrastructure
19.
Hum Reprod ; 21(7): 1771-6, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16549422

ABSTRACT

BACKGROUND: Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. METHODS: Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. RESULTS: Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). CONCLUSIONS: Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes.


Subject(s)
Chromosomes, Human/ultrastructure , Cryopreservation/methods , Oocytes/drug effects , Sucrose/pharmacology , Female , Humans , Mitosis/physiology , Oocytes/physiology , Oocytes/ultrastructure , Spindle Apparatus/ultrastructure , Sucrose/administration & dosage
20.
Mol Reprod Dev ; 72(2): 161-70, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16007680

ABSTRACT

Demecolcine-induced enucleation (IE) of mouse oocytes has been shown to improve development to term of cloned mice. In this study, we characterized the kinetics and morphological progression of bovine oocytes subjected to IE, and evaluated their ability to support embryo development to the blastocyst stage after nuclear transfer (NT). In vitro matured bovine oocytes were parthenogenetically activated and subsequently exposed to demecolcine at various times post-activation. Onset and duration of demecolcine treatment significantly altered activation and IE frequencies, which varied from 7.1% to 100% and 33.3% to 91.7%, respectively, at 5 hr post-activation. A significant decrease in IE frequencies was observed at 17 hr post-activation (3.4%-46.1%), possibly due to reincorporation of chromosomes into the oocyte after incomplete second polar body (PB) extrusion. Oocytes were reconstructed by NT before (treatment 1) or after (treatment 2) activation and demecolcine treatment, and cultured in vitro. Cleavage (48.1%-54.2%) and blastocyst rates (15.7%-19%) were equivalent for the two treatments, as well as the total cell number in NT blastocysts. Furthermore, most of the blastocysts were completely diploid (treatment 2) or heteroploid but with a majority of diploid nuclei (treatment 1). Our results demonstrate that the IE method can be successfully used to produce enucleated bovine cytoplasts that are competent to support development to the blastocyst stage after NT. This technically simple approach may provide a more efficient method to enhance the success rate of NT procedures. Further studies are needed to improve the in vitro development efficiency and to expand our understanding of the mechanism(s) involved in demecolcine-induced enucleation.


Subject(s)
Cell Nucleus/drug effects , Cytoplasm/physiology , Demecolcine/pharmacology , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Nuclear Transfer Techniques , Animals , Cattle , Cells, Cultured , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Karyotyping , Meiosis/drug effects , Microscopy, Confocal , Oocytes/cytology , Oocytes/drug effects , Ploidies
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