以immunocytochemistry 顯影,  laser confocal microscopy偵測卵子纺綞體&染色體
高齡卵子纺綞體&染色體異常率明顯較高

Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2007 Feb;24(1):6-9.

[Influence of maternal age on meiotic spindle and chromosome configuration of oocytes].

[Article in Chinese]

Chen W1, Hu J, Liu Q, Wei YL, Zhu GJ.

Author information

Abstract

OBJECTIVE:

To study the effect of maternal age on meiotic spindle and chromosome configuration of oocytes.

METHODS:

Spindle and chromosome configuration was examined in day 1 unfertilized human oocytes after in vitro fertilization (IVF) andintracytoplasmic sperm injection(ICSI) by immunocytochemistry and visualized by laser confocal microscopy.

RESULTS:

Statistically significant differences were observed on normal spindle and chromosome configurations of oocytes between 25-29 maternal age group (33% and 31%, respectively), and 30-34 age group (P< 0.05) as well as 35-40 age group(0%, P<0.01). The incidence of abnormal spindle and chromosome configurations of oocytes from 30-34 and 35-40 maternal age groups was much higher than that of oocytes from 25-29 age group (P<0.01, P<0.05).

CONCLUSION:

Incidence of abnormal spindle and chromosome configuration of oocytes is related to maternal age. It could be an important reason of age related oocyte aneuploidy.

雷射輔助ICSI,打薄ZP70%再ICSI, 可能可減少卵子受損率, 提高胚胎囊胚率, 著床率

Clin Exp Reprod Med. 2011 Sep;38(3):148-52. doi: 10.5653/cerm.2011.38.3.148. Epub 2011 Sep 30.

Efficiency of laser-assisted intracytoplasmic sperm injection in a human assisted reproductive techniques program.

Choi KH1, Lee JH, Yang YH, Yoon TK, Lee DR, Lee WS.

Author information

Abstract

OBJECTIVE:

Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics.

METHODS:

Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LA-ICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics.

RESULTS:

Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients.

CONCLUSION:

LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.

無精症睪丸切片TESE後完全不動精蟲可經由4方法挑選具生命力精蟲
(1)半滲透壓溶劑---活精蟲尾部呈現捲曲
(2)化學藥劑刺激---活精蟲尾部呈現振動
(3)精蟲尾部彎曲性---活精蟲頭部尾部長軸呈現不同方向
(1)精蟲尾部雷射反應---活精蟲尾部單發雷射後呈現捲曲

Andrology. 2015 Mar;3(2):156-62. doi: 10.1111/andr.286. Epub 2014 Oct 20.

How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view.

Nordhoff V1.

Author information

Abstract

A viable spermatozoon is the prerequisite for initiating fertilization in intracytoplasmic sperm injection. Usually motility is the primary sign used to determine a sperm's viability. However, in every in vitro fertilization (IVF) laboratory cases are observed in which none or only few spermatozoa are motile. This can occur in treatment cycles involving ejaculated samples but is most common in cases where surgically extracted testicular spermatozoa are used. To aid in selection, several techniques have been developed to identify viable spermatozoa from the immotile fraction. Amongst the more commonly used approaches are (i) the hypo-osmotic swelling test (ii) chemical substances for induction of tail movements (iii) the sperm tail flexibility test and (iv) laser-assisted immotile sperm selection. All can be used routinely in an IVF laboratory with each having both strengths and weaknesses. It is the purpose of this short review to focus on the technical issues involved in the performance of each of these techniques and to highlight the advantages and disadvantages of each approach.

© 2014 American Society of Andrology and European Academy of Andrology.

901高齡(40-46歲)IVF病患顯示，植入3胚胎懷孕率分別為
40歲: 25 %
41歲: 20 %
42歲: 16 %
43歲: 17 %
44歲: 8 % 
45歲: 6 % 
46歲: 0 %

44歲以上無人活產

J Assist Reprod Genet. 2016 Jun 1. [Epub ahead of print]

The effect on pregnancy and multiples of transferring 1-3 embryos in women at least 40 years old.

Alasmari NM1, Son WY1, Dahan MH2.

Author information

Abstract

PURPOSE "CAPSULE" IS MANDATORY. PLEASE PROVIDE.SINGLE EMRBYO TRANSFER (SET) IN WOMEN ≥40 YEARS OLD APPEARS TO LOWER THE CHANCE OF A PREGNANCY. HOWEVER, IT MINIMIZES THE RISK OF MULTIPLE PREGNANCIES EVEN IN WOMEN OF ADVANCED MATERNAL AGE. THEREFORE, WOMEN 40 YEARS OF AGE OR OLDER SHOULD BE OFFERED (SET).: This study was performed to investigate the multiple pregnancies and live birth rates when 1-3 embryos are transferred at this age in women at least 40 years of age.

METHOD:

A retrospective analysis of data which included 631 women aged 40 to 46 years, who underwent 901 cycles of IVF, from August 2010 to June 2012 was undertaken. These women underwent embryo transfer of 1-3 non-donor fresh embryo(s).

RESULTS:

Results suggested that the average pregnancy rate when up to three embryos were transferred was 25 % for women 40 years old, 20 % for women 41 years old, 16 % for women 42 years old, 17 % for women 43 years old, 8 % for women 44 years old, 6 % for women 45 years old, and 0 % for women 46 years old. No live births occurred in women treated after their 44th birthday, and only patients younger than 42 years of age receiving double embryo transfer had a live birth of twins. Live birth rates increased as more embryos were transferred for 40- and 42-year-old subjects (p = 0.01 and 0.05, respectively).

CONCLUSIONS:

From these results, it was concluded that SET in women ≥40 years old appears to lower the chance of a pregnancy. However, it minimizes the risk of multiple pregnancies even in women of advanced maternal age. Women 40 years of age or older should be offered single-embryo transfer. Further studies are needed to determine risk of multiple pregnancies in women 42 years of age or older when few embryos are transferred. Decisions on the number of embryos to transfer should be on a case by case basis, in discussion with the patient.

施打破卵針當天P4過高(>1.5)只影響當期子宮內膜品質
不影響卵子品質&胚胎品質
不影響下一週期子宮內膜品質
不影響下一週期FET懷孕率

J Assist Reprod Genet. 2016 Jun 4. [Epub ahead of print]

Is it the egg or the endometrium? Elevated progesterone on day of trigger is not associated with embryo ploidy nor decreased success rates in subsequent embryo transfer cycles.

Kofinas JD1, Mehr H2, Ganguly N2, Biley Y2, Bochkovsky S2, McCulloh D2, Grifo J2.

Author information

Abstract

PURPOSE:

The purpose of our study was to determine if progesterone (P4) values on day of trigger affect certain cycle outcome parameters, ploidy status of embryos, as well as pregnancy outcomes in the subsequent first frozen embryo transfer cycle.

METHODS:

Two hundred thirty-eight patients undergoing pre-gestational screening and freeze all protocol at our fertility center from 2013 to 2014 were included. Excluded patients were those whom had cancelled cycles prior to egg retrieval as well as cycles utilizing donor eggs. Once patients were identified as eligible for this study, frozen serum from the day of trigger was identified and analyzed using the Siemens Immulite 2000. Number of eggs retrieved, number of available embryos for biopsy, and number of euploid/aneuploid embryos were analyzed. The first frozen embryo transfer cycle was linked to the initial egg retrieval and outcomes including pregnancy rates, and live birth/ongoing pregnancy rates were calculated and analyzed. A discriminatory P4 value of 1.5 ng/ml was set. Group A had P4 values of less than 1.5 ng/ml and group B had P4 values greater than or equal to 1.5 ng/ml. T tests and chi-squared tests were used for statistical analysis.

RESULTS:

Group A had an average trigger P4 value of 0.87 +/- 0.3 and group B had an average trigger P4 of 2.1 +/- 0.8. Table 1 shows the baseline characteristics of both group A and group B. The only significant difference between the two groups was total gonadotropin dosage (IU) with a p value of 0.02 and estradiol (pg/ml) at trigger, also with a p value of 0.02 (Table 1). Number of eggs retrieved, number of embryos biopsied, number euploid/aneuploid, and non-diagnosis embryos were all non-significant. Chi-square analysis was used to compare pregnancy rates between the two groups after the first frozen embryo transfer cycle. Group A had a pregnancy rate of 72 % and Group B had a pregnancy rate of 66.7 %, which was not significant. Ongoing pregnancy/live birth rates were 65.6 % in group A and 66.67 % in group B, also not significant (Table 2).

CONCLUSIONS:

P4 values on day of trigger do not affect number of eggs retrieved and number of chromosomally normal embryos available for transfer in a subsequent embryo transfer cycle. Elevated P4 values (≥1.5 ng/ml) also do not affect pregnancy rates or live birth/ongoing pregnancy rates in the first subsequent frozen embryo transfer cycle.

IVF卵子未受孕再施與ICSI或
不成熟卵子經由體外成熟培養IVM再施與ICSI
可達49%卵子受孕率,
可達14.7%囊胚成長率

J Assist Reprod Genet. 2016 Jun 2. [Epub ahead of print]

Delayed intracytoplasmic sperm injection (ICSI) with trophectoderm biopsy and preimplantation genetic screening (PGS) show increased aneuploidy rates but can lead to live births with single thawed euploid embryo transfer (STEET).

Sachdev NM1, Grifo JA2, Licciardi F2.

Author information

Abstract

PURPOSE:

The aim of this study was to report the results of IVF with trophectoderm biopsy and preimplantation genetic screening (PGS) following delayed intracytoplasmic sperm injection (ICSI).

METHODS:

Patients undergoing IVF with PGS and delayed ICSI were included in the study. Indications for delayed ICSI included absent or poor fertilization via standard insemination or more than 50 % immature oocytes, noted post-cumulus stripping for standard ICSI procedure. Delayed ICSI was performed the day after retrieval due to absent or poor fertilization. The immature oocytes were kept in extended culture, and if demonstrated maturity, ICSI was performed. Primary outcome included fertilization rate and blastocyst stage formation, defined by the number of blastocysts for biopsy. Secondary outcome included aneuploidy rate and pregnancy outcomes following single thawed euploid embryo transfers (STEET).

RESULTS:

Sixteen patients with delayed ICSI were included in the study. Twelve were due to poor fertilization and four secondary to immature oocytes. A total of 219 oocytes were retrieved; ten were frozen upon patient request, 168 had standard insemination, and 13 had routine ICSI on the day of retrieval. A total of 129 oocytes underwent delayed ICSI. Sixty-three (49 %) fertilized, 19 (14.7 %) reached blastocysts for biopsy; fivw of which were chromosomally normal (26.3 %). Three patients underwent STEET of a delayed ICSI embryo; all three resulted in live births, including one embryo biopsied on day 8 of development.

CONCLUSION:

Fertilization failure or an excessive proportion of immature oocytes in an IVF cycle, necessitating delayed ICSI, showed equivalent fertilization and blast formation rates. With the implementation of trophectoderm biopsy and PGS, these embryos can lead to healthy live born babies.

排卵日後14天驗hCG(非植入後14天), 若hCG>100活產率大
排卵日後12天驗hCG, 若hCG>40活產率大

J Assist Reprod Genet. 2016 Jun 4. [Epub ahead of print]

Serum hCG-β levels of postovulatory day 12 and 14 with the sequential application of hCG-β fold change significantly increased predictability of pregnancy outcome after IVF-ET cycle.

Sung N1,2, Kwak-Kim J1, Koo HS2, Yang KM3.

Author information

Abstract

PURPOSE:

To investigate hCG-β level on postovulatory day (POD) 12 and its fold increase as predictors for pregnancy outcome after in vitro fertilization (IVF) cycles.

METHODS:

A retrospective cohort study was performed in total 1408 fresh and 598 frozen cycles between November 2008 and October 2011, which resulted in biochemical pregnancy, early pregnancy loss, or live birth of singleton pregnancy. The serum hCG-β levels of POD 12 and 14 were compared among biochemical pregnancy, early pregnancy loss, and live birth groups. The cutoff values of POD 12 and 14 hCG-β levels and the degree of hCG-β increase from POD 12 to 14 were determined for each pregnancy outcome.

RESULTS:

POD 12 and 14 hCG-β levels stratified based on pregnancy outcomes were significantly different among the biochemical pregnancy, early pregnancy loss, and live birth in both fresh and frozen cycles. Serum hCG-β levels of POD 12 and 14 and the fold increase of hCG-β levels from POD 12 to 14 significantly predict pregnancy outcomes after fresh and frozen cycles. Among these, the cutoff value of POD 14 hCG-β had the highest sensitivity and positive predictive value (PPV). In fresh cycles, the cutoff values of POD 12 and 14 serum hCG-β levels for clinical pregnancies were 30.2 mIU/mL (sensitivity 81.3 %, specificity 79.6 %, and PPV 92.3 %) and 70.5 mIU/mL (sensitivity 88.4 %, specificity 85.2 %, and PPV 94.7 %). In pregnancies with POD 12 serum hCG-β levels ≥30.2 mIU/mL, the cutoff level of increase of hCG-β for clinical pregnancy was 2.56 (sensitivity 73.6 %, specificity 72.4 %, and PPV 97.8 %). Sequential application of cutoff values such as POD 12 hCG-β and fold increase of hCG-β improved predictability of pregnancy outcome as compared with that of POD 12 hCG-β alone. The cutoff values of POD 12 and 14 serum hCG-β levels for live birth were 40.5 mIU/mL (sensitivity 75.2 %, specificity 72.6 %, PPV 78.9 %) and 104.5 mIU/mL (sensitivity 80.3 %, specificity 74.1 %, PPV 80.8 %). In the frozen cycles, the cutoff values of POD 12 and 14 serum hCG-β level for clinical pregnancy were 31.5 IU/L (sensitivity 80.4 %, specificity 71.1 % and PPV 90 %) and 43.5 mIU/mL (sensitivity 72.6 %, specificity 71.7 %, PPV 77.2 %). In pregnancies with POD 12 serum hCG-β level ≥31.5 mIU/mL, the cutoff value for fold increase of hCG-β was 2.38 for clinical pregnancy (sensitivity 81.6 %, specificity 71.4 % and PPV 87.9 %). The cutoff values of POD 12 and 14 for live birth were 43.5 mIU/mL (sensitivity 72.6 %, specificity 71.7 %, PPV 77.2 %) and 101.6 mIU/mL (sensitivity 79.6 %, specificity 71.1 %, PPV 78.4 %). Sequential application of cutoff values for POD 12 hCG-β level and fold increase of hCG-β significantly increased PPV for live birth but not clinical pregnancy in frozen cycles.

CONCLUSIONS:

Early prediction of pregnancy outcome by using POD 12 and 14 cutoff levels and sequential application of cutoff value of fold increase could provide appropriate reference to health care providers to initiate earlier management of high-risk pregnancies and precise follow-up of abnormal pregnancies.

動物研究顯示卵巢冷凍解凍移植後，卵巢受損主因在卵巢移植過程循環下降缺血造成卵巢功能下降

Hum Reprod. 2016 Jun 16. pii: dew144. [Epub ahead of print]

Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury.

Lee J1, Kong HS1, Kim EJ1, Youm HW2, Lee JR3, Suh CS1, Kim SH4.

Author information

Abstract

STUDY QUESTION:

What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury?

SUMMARY ANSWER:

Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function.

WHAT IS KNOWN ALREADY:

During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve.

STUDY DESIGN, SIZE, DURATION:

A total of 80 B6D2F1 female mice were randomly allocated to 2 control and 6 experimental groups according to the presence or the absence of transplantation (n = 10/group). The control groups consisted of fresh or vitrified-warmed controls that had the whole ovary fixed without transplantation (fresh and vitri-con, respectively). The experimental groups were further divided according to the presence of vitrification (fresh or vitrified-warmed) and the transplantation period (2 [D2], 7 [D7] or 21 [D21] days).

PARTICIPANTS/MATERIALS, SETTING, METHODS:

In the control groups, fresh and vitrified-warmed ovaries were immediately fixed after the collection (fresh) and the vitrification-warming process (vitrification control, vitri-con), respectively. Of those experimental groups, three were auto-transplanted with fresh whole ovary (FrOT; FrOT-D2, FrOT-D7 and FrOT-D21). For the other three groups, the ovaries were harvested and stored in liquid nitrogen for 1 week after vitrification and then warmed to auto-transplant the vitrified whole ovaries (vitrified ovary [VtOT]; VtOT-D2, VtOT-D7 and VtOT-D21). After 2, 7 or 21 days of grafting, the grafts and blood sera were collected for analysis by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, CD31 immunohistochemistry and follicle-stimulating hormone enzyme-linked immunosorbent assay.

MAIN RESULTS AND THE ROLE OF CHANCE:

The vitrification-warming procedure decreased the proportion of intact follicles (Grade 1, G1) (vitri-con 50.3% versus fresh 64.2%) but there was a larger decrease due to ischemic injury after transplantation (FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovary group compared with the fresh control, but it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0% and FrOT-D2: 26.8%). The mean number of follicles per section and percentage of CD31-positive area significantly decreased after vitrification but decreased to a larger extent after transplantation (number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, FrOT-D2: 17.9 ± 2.1; CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9% and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1% and FrOT-D21: 70.7%; CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6% and FrOT-D21: 7.5 ± 0.8%). Although there was no significant difference between the two transplantation groups at each evaluation, the serum follicle-stimulating hormone level of both groups significantly decreased over time.

蛋白質體研究顯示，副睪細精管在精子成熟過程中伴演重要角色
非僅僅輸送儲存精蟲管道角色

Hum Reprod Update. 2016 Jun 15. [Epub ahead of print]

The human epididymis: its function in sperm maturation.

Sullivan R1, Mieusset R2.

Author information

Abstract

BACKGROUND:

Spermatozoa acquire their fertilizing ability and forward motility properties during epididymal transit. Our knowledge of gamete physiology is based on studies conducted in laboratory and domestic species; our knowledge of these processes in humans is limited. Medical indications for assisted reproductive technologies (ART) have progressed to include male infertility. Surgical procedures allow collection of spermatozoa from all along the human excurrent ducts, and the former have been used with some success in reproductive medicine. This has raised questions over the role of the epididymis in human sperm physiology.

OBJECTIVE AND RATIONALE:

To reanalyze what we now know about epididymal physiology in humans and to assess the relevance of laboratory animal models for understanding human physiology and the pathophysiology of the epididymis.

SEARCH METHODS:

A systematic bibliographic search of PubMed for articles published in English before May 2015 was carried out using the search terms 'epididymis' and 'sperm maturation'. Literature on the consequences of vasectomy on the epididymis was also searched.

OUTCOMES:

Whereas the proximal epididymis is almost exclusively occupied by efferent ducts, the sperm reservoir capacity is poorly developed in humans. At the molecular level, the human transcriptome and proteome show some segment specificity; conflicting results persist with regard to secretome variation along the tubule. The number of genes regulated along the excurrent ducts in men is lower when compared to rodent species, but remains significant. It is challenging to reconcile biochemical and physiological studies with clinical data obtained from men undergoing reanastomosis of the vas deferens at different points along the excurrent duct. We propose that vasectomy/vasovasostomy is a model to understand the consequences of obstruction on epididymis function in humans.

WIDER IMPLICATIONS:

Despite the scarcity of biological material available, the interspecies variability of the male reproductive tract urges us to use modern molecular and cellular biology tools to better understand human epididymis physiology in order to apply ART in a more responsible manner.

PCO多囊性卵巢表現症狀有3
A.月經異常ovulatory dysfunction
B. 男性賀爾蒙過高hyperandrogenism
C. 超音波顯示卵巢多囊PCOM 

區分為4型
A+B+C: hyperandrogenism + ovulatory dysfunction+PCOM
A+B: hyperandrogenism + ovulatory dysfunction
B+C: hyperandrogenism + PCOM
A+C: ovulatory dysfunction + PCOM 

Fertil Steril. 2016 May 26. pii: S0015-0282(16)61268-2. doi: 10.1016/j.fertnstert.2016.05.009. [Epub ahead of print]

Introduction: Determinants of polycystic ovary syndrome.

Azziz R1.

Author information

Abstract

The polycystic ovary syndrome (PCOS) is recognized as one of the most common endocrine abnormalities of humans, with global prevalences so far generally 5%-15%. Overall, the disorder appears to be an ancient complex genetic trait, perhaps dating at least 50,000 years ago. The phenotype of PCOS can be subdivided into four different types. Phenotype A and B (hyperandrogenism + ovulatory dysfunction, with [A] and without [B] polycystic ovarian morphology [PCOM], respectively) can be considered to represent the "classic" form of the disorder. Phenotype C is the so-called "ovulatory" PCOS (hyperandrogenism + PCOM only). And phenotype D is often referred to as "nonhyperandrogenic" PCOS (ovulatory dysfunction + PCOM only). The different phenotypes vary in the degree to which they are associated with an increased risk for metabolic dysfunction and reproductive complications. There are a number of determinants of the epidemiology (prevalence) and presentation (phenotype) of PCOS, including environmental (e.g., socioeconomic, geographic, toxicologic, life-style, and dietary) and genetic (e.g., gene variants, epigenetic, and race/ethnicity) factors. Finally a better understanding of the evolutionary determinants of PCOS has the potential for providing additional insight into those factors determining the etiology, prevalence, and persistence of a disorder that appears to be, superficially at least, an evolutionary paradox.

將羊水細胞分離出幹細胞(pluripotency of human amniotic fluid stem cells (hAFSCs)[含stem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA)]
將幹細胞與豬濾泡液共同培養7-10天, 約2%幹細胞可形成類似原始卵細胞

Biol Reprod. 2014 Apr 3;90(4):73. doi: 10.1095/biolreprod.113.112920. Print 2014 Apr.

Human amniotic fluid stem cells possess the potential to differentiate into primordial follicle oocytes in vitro.

Yu X1, Wang N, Qiang R, Wan Q, Qin M, Chen S, Wang H.

Author information

Abstract

Previous reports have demonstrated that embryonic stem cells were capable of differentiating into primordial germ cells through the formation of embryoid bodies that subsequently generated oocyte-like cells (OLCs). Such a process could facilitate studies of primordial follicle oocyte development in vitro and regenerative medicine. To investigate the pluripotency of human amniotic fluid stem cells (hAFSCs) and their ability to differentiate into germ cells, we isolated a CD117(+)/CD44(+) hAFSC line that showed fibroblastoid morphology and intrinsically expressed bothstem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA). To encourage differentiation into OLCs, the hAFSCs were first cultured in a medium supplemented with 5% porcine follicular fluid for 10 days. During the induction period, cell aggregates formed and syntheses of steroid hormones were detected; some OLCs and granulosa cell-like cells could be loosened from the surface of the culture dish. Cellaggregates were collected and replated in oocyte culture medium for an additional 7-10 days. OLCs ranging from 50 to 120 μm presenting zona pellucida were observed in cumulus-oocyte complexes; some OLCs developed spontaneously into multicell structures similar to preimplantation embryos. Approximately 2% of the hAFSCs differentiated to meiotic germ cells that expressed folliculogenesis- and oogenesis-associated markers. Although the in vitro maturation and fertilization potentials are as yet unproven, short-term (<25 days) and high-efficiency (>2%) derivation of OLCs from hAFSCs might provide a new approach to the study of human germ cell development in vitro.

培養液ammonium來緣: 培養液內之glutamine & 培養液蛋白添加劑
培養液置於37度C下4天， ammonium會增加至損害胚胎程度
培養液置於2-8度C下6週，ammonium會增加至損害胚胎程度

Hum Reprod. 2016 Jun;31(6):1192-9. doi: 10.1093/humrep/dew059. Epub 2016 Apr 6.

Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.

Kleijkers SH1, van Montfoort AP1, Bekers O2, Coonen E1, Derhaag JG1, Evers JL1, Dumoulin JC3.

Author information

Abstract

STUDY QUESTION:

Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation?

SUMMARY ANSWER:

Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C.

WHAT IS KNOWN ALREADY:

Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice.

STUDY DESIGN, SIZE, DURATION:

Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements.

PARTICIPANTS/MATERIALS, SETTING, METHODS:

Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days.

MAIN RESULTS AND THE ROLE OF CHANCE:

All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet.

胚胎植入前雌激素補充劑量(增加型, 固定型), 二者懷孕率無明顯差異
(1)增加型 increasing E dose (ID) (oral: 2 mg/day day(d)1-7, 4 mg days d8-12, 6 mg d13-embryo transfer; transdermal: 75 µg/3 days on d1-6, 150 µg/3 days d7-embryo transfer)
(2) 固定型 constant dose (CD) of estrogen (oral: 6 mg/day 1-embryo transfer; transdermal: 150 µg/3 days d1-embryo transfer)

Hum Reprod. 2016 May 1. pii: dew099. [Epub ahead of print]

Endometrial preparation: effect of estrogen dose and administration route on reproductive outcomes in oocyte donation cycles with fresh embryo transfer.

Madero S1, Rodriguez A1, Vassena R2, Vernaeve V1.

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Abstract

STUDY QUESTION:

Is there a difference in live birth rates following endometrial preparation with either a constant or increasing estrogen dose in fresh embryo transfer from oocyte donation cycles?

SUMMARY ANSWER:

There is no difference in live birth rates between a constant dose versus an increasing dose of estrogen after fresh embryo transfer in oocyte donation cycles with oral or transdermal supplementation.

WHAT IS KNOWN ALREADY:

Endometrial preparation (EP) with estrogen and progesterone, and embryo-endometrial synchronicity are determinant for adequate embryo implantation. Estrogen is crucial and different exogenous administration patterns could imply variations on EP. Moreover, estrogen undergoes metabolization by the intestines and liver when administered orally, an effect that is bypassed by transdermal administration. Information on the effect of replacement patterns and route of administration of E on reproductive outcomes of women undergoing fresh embryo transfer from oocyte donation cycles is scarce.

STUDY DESIGN, SIZE, DURATION:

Retrospective cohort study including 8362 embryo transfers following ICSI, corresponding to 8254 patients, between October 2010 and March 2015. A total of 5593 (66.9%) patients received an increasing E dose (ID) (oral: 2 mg/day day(d)1-7, 4 mg days d8-12, 6 mg d13-embryo transfer; transdermal: 75 µg/3 days on d1-6, 150 µg/3 days d7-embryo transfer) while 2769 (33.1%) received a constant dose (CD) of estrogen (oral: 6 mg/day 1-embryo transfer; transdermal: 150 µg/3 days d1-embryo transfer). Embryos were generated by ICSI with fresh or vitrified donor oocytes fertilized with either fresh or frozen sperm from either the couple partner or donor.

PARTICIPANTS/MATERIALS, SETTING, METHODS:

Cohort allocation was not related to patient characteristics; instead it reflected an internal policy change in E administration. Effect of estrogen dose (ID versus CD) on biochemical, clinical, ongoing and live birth rates, stratified by administration route, was analyzed by univariate and multivariate analysis adjusted by donor and recipient demographic and cycle characteristics.

MAIN RESULTS AND THE ROLE OF CHANCE:

No difference in live birth rate was found between CD and ID for oral (33.0 versus 32.5%, P = 0.81) and transdermal (35.3 versus 33.5%, P = 0.33) supplementation. Biochemical pregnancy rate was higher in CD than ID (53.7 versus 47.5%, P < 0.001) when patients received oral supplementation. Adjusted analysis confirmed that oral administration had a greater impact on biochemical pregnancy rates than transdermal (odds ratio (OR) 1.28; 95% confidence interval (CI) 1.11-1.48, P = 0.001 versus OR 1.13; 95% CI 1.00-1.30, P = 0.055). Sub-analysis of transfers between day 12 and 15 of estrogen supplementation showed no difference between CD and ID in pregnancy outcomes. Demographic variables and cycle characteristics were comparable between both groups. Moreover, the use of the oocyte donation model reduces confounding factors related to oocyte age, embryo aneuploidy, and embryo quality.

2-cell階段胚胎高達43.2%胚胎具多核性胚葉細胞
2-cell階段多核性胚胎大部分具有自我修復能力
懷孕率類似一般單核性胚胎

Fertil Steril. 2016 May 18. pii: S0015-0282(16)61138-X. doi: 10.1016/j.fertnstert.2016.04.041. [Epub ahead of print]

Impact of multinucleated blastomeres on embryo developmental competence, morphokinetics, and aneuploidy.

Balakier H1, Sojecki A2, Motamedi G2, Librach C3.

Author information

Abstract

OBJECTIVE:

To study the effect of human embryo multinucleation on the rate of aneuploidy, in vitro developmental morphokinetics, and pregnancy outcome.

DESIGN:

Retrospective study.

SETTING:

University-affiliated fertility center.

PATIENT(S):

A total of 296 patients undergoing IVF cycles.

INTERVENTION(S):

None.

MAIN OUTCOME MEASURE(S):

Rate of multinucleation at the 2- and 4-cell stage, time-lapse morphokinetic parameters from zygote to blastocyst stage, ploidy of embryos analyzed by means of trophectoderm biopsy and array comparative genomic hybridization (PGS), and pregnancy outcome.

RESULT(S):

A total of 1,055 out of 2,441 (43.2%) embryos evaluated with the use of the Embryoscope time-lapse system showed blastomere multinucleation at the 2-cell stage (MN2). The frequency of this abnormality was substantially reduced in 4-cell-embryos (15.0%). Among all clinical factors analyzed, only maternal age had a positive correlation with multinucleation rate. The timing of cleavage divisions from the pronuclear fading to 5-cell embryo was significantly longer (1.0-2.5 h) in MN2 embryos than in non-MN2 control samples. Of the total embryos tested with the use of PGS (n = 607), the rates of multinucleation were similar in euploid versus aneuploid blastocysts (40.8% and 46.7%, respectively). All 24 chromosomes contributed to aneuploidy of MN2 embryos. There were 61 transfers of MN2 embryos that resulted in 45.9% clinical pregnancies and a 31.6% implantation rate.

CONCLUSION(S):

The frequency of multinucleation is high in human embryos cultured in vitro and equally affects euploid and aneuploid human embryos. It appears that most MN embryos have the capacity for self-correction during early cleavage divisions and can develop into euploid blastocysts resulting in healthy babies.