The relationship between anti-Mu¨ llerian hormone, androgen
and insulin resistance on the number of antral follicles
in women with polycystic ovary syndrome
Mei-Jou Chen1,2, Wei-Shiung Yang2,3, Chi-Ling Chen2, Ming-Yih Wu1, Yu-Shih Yang1
and Hong-Nerng Ho1,4
1Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100, Taiwan;
2Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; 3Department of Internal
Medicine, National Taiwan University Hospital, Taipei, Taiwan
4Correspondence address. Tel: þ886-2-2356-2175; Fax: þ886-2-2341-8557; E-mail: hnho@ntu.edu.tw
BACKGROUND: Anti-Mu¨ llerian hormone (AMH) is a biomarker that predicts the number of antral follicles and is
involved in follicle arrest for women with polycystic ovary syndrome (PCOS). We investigated the association between
the characteristic hyperandrogenemia, insulin resistance (IR), AMH, and the morphology and size of ovaries for
women with PCOS. METHODS: A total of 99 Taiwanese women with PCOS who were willing to undergo vaginal
ultrasonography were enrolled in this cross-sectional study. RESULTS: The number of antral follicles and the
ovarian volume showed a significant correlation with AMH, total testosterone and the free androgen index, but not
with age, body mass index (BMI) or the homeostasis model assessment of insulin resistance (HOMA-IR). AMH
had a significant negative association with both BMI and HOMA-IR. Multiple stepwise regression analysis demonstrated
that AMH, BMI and total testosterone were independently related to the number of antral follicles.
AMH and total testosterone were the main determinants for ovarian volume in a stepwise regression model.
CONCLUSIONS: Our results suggest that not only the AMH level, but also obesity, IR and elevated androgen
levels may relate to the development of the large size of antral follicle pool and ovarian volume in women with
PCOS. Obesity and IR may enhance the follicular excess through the dysregulation of AMH or through the
pathway of hyperandrogenemia. These findings might partly explain why adequate body weight management and
improvement in IR can improve the ovulatory function for women with PCOS.
Keywords: polycystic ovary syndrome; anti-Mu¨llerian hormone; obesity; insulin resistance; antral follicle count

Impaired insulin-dependent glucose metabolism in granulosa-lutein cells from anovulatory women with polycystic ovaries

S. Rice^1,4, N. Christoforidis², C. Gadd², D. Nikolaou², L. Seyani³, A. Donaldson³, R. Margara², K. Hardy¹ and S. Franks^1,5

¹ Institute of Reproductive and Developmental Biology, ² Department of Obstetrics and Gynaecology, Imperial College London, London and ³ Clinical Chemistry Laboratory, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

⁵ To whom correspondence should be addressed. Email: s.franks@imperial.ac.uk

BACKGROUND: Insulin resistance and hyperinsulinaemia are well-recognized characteristics of anovulatory women with polycystic ovary syndrome (PCOS) but, paradoxically, steroidogenesis by PCOS granulosa cells remains responsive to insulin. The hypothesis to be tested in this study is that insulin resistance in the ovary is confined to the metabolic effects of insulin (i.e. glucose uptake and metabolism), whereas the steroidogenic action of insulin remains intact. METHODS: Granulosa-lutein cells were obtained during IVF cycles from seven women with normal ovaries, six ovulatory women with PCO (ovPCO) and seven anovulatory women with PCO (anovPCO). Mean body mass index was in the normal range in all three groups. Granulosa-lutein cells were cultured with insulin (1, 10, 100 and 1000 ng/ml) and LH (1, 2.5 and 5 ng/ml). Media were sampled at 24 and 48 h and analysed for glucose uptake, lactate production and (48 h only) progesterone production. RESULTS: Insulin-stimulated glucose uptake by cells from anovPCO was attenuated at higher doses of insulin (100 and 1000 ng/ml) compared with that by cells from either ovPCO (P=0.02) or controls (P=0.02). Insulin and LH stimulated lactate production in a dose-dependent manner, but insulin-dependent lactate production was markedly impaired in granulosa-lutein cells from anovPCO compared with either normal (P=0.002) or ovPCO (P<0.0001). By contrast, there was no difference in insulin-stimulated progesterone production between granulosa-lutein cells from the three ovarian types. CONCLUSIONS: Granulosa-lutein cells from women with anovPCOS are relatively resistant to the effects of insulin-stimulated glucose uptake and utilization compared with those from normal and ovPCO, whilst maintaining normal steroidogenic output in response to physiological doses of insulin. These studies support the probability of a post-receptor, signalling pathway-specific impairment of insulin action in PCOS.

Key words: granulosa cells/insulin resistance/LH/polycystic ovaries/progesterone

⁴ Present address: Department of Basic Medical Sciences, St George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 0RE, UK

The role of metformin in polycystic ovary syndrome: a systematic review

Etelka Moll¹, Fulco van der Veen and Madelon van Wely

Centre for Reproductive Medicine, Department of Obstetrics and Gynaecology, Academic Medical Centre, PO Box 22700, 1100 DE, Amsterdam, The Netherlands

¹ Correspondence address. Tel: +31-20-5663557; Fax: +31-20-6963489; E-mail: e.moll@amc.uva.nl

This meta-analysis evaluated the effectiveness of metformin in subfertile women with polycystic ovary syndrome (PCOS). Only randomized trials investigating the effectiveness of metformin and PCOS definition consistent with the Rotterdam consensus criteria, were eligible. Primary outcome was live birth rate. A literature search identified 27 trials. In therapy naïve women, we found no evidence of a difference in live birth rate when comparing metformin with clomifene citrate (CC) [relative risks (RR) 0.73; 95% confidence interval (CI) 0.51–1.1] or comparing metformin plus CC with CC (RR 1.0; 95% CI 0.82–1.3). In CC-resistant women, metformin plus CC led to higher live birth rates than CC alone (RR 6.4; 95% CI 1.2–35); metformin also led to higher live birth rates than laparoscopic ovarian drilling (LOD) (RR 1.6; 95% CI 1.1–2.5). We found no evidence for a positive effect of metformin on live birth when added to LOD (RR 1.3; 95% CI 0.39–4.0) or FSH (RR 1.6; 95% CI 0.95–2.9), or when co-administered in IVF (RR 1.5; 95% CI 0.92–2.5). In IVF, metformin led to fewer cases of ovarian hyperstimulation syndrome (OHSS) (RR 0.33; 95% CI 0.13–0.80). This meta-analysis demonstrates that CC is still first choice therapy for women with therapy naïve PCOS. In CC-resistant women, the combination of CC plus metformin is the preferred treatment option before starting with LOD or FSH. At present, there is no evidence of an improvement in live birth when adding metformin to LOD or FSH. In IVF, metformin leads to a reduced risk of OHSS.

Key words: infertility / metformin / PCOS / pregnancy / review

Received on April 12, 2007; revised June 7, 2007; accepted on June 29, 2007

Richard Fleming, Ph.D.,a Lyndal Harborne, B.Med.,a David T. MacLaughlin, Ph.D.,b
Daniel Ling,b Jane Norman, M.D.,a Naveed Sattar, M.R.C.Path.,a and David B. Seifer, M.D.c
a University Department of Obstetrics and Gynaecology, Royal Infirmary, Glasgow, United Kingdom; b Paediatric Research
Laboratory, Massachusetts General Hospital for Children and Harvard Medical School, Boston, Massachusetts; and c Department
of Obstetrics, Gynecology and Reproductive Sciences, Division of Reproductive Endocrinology and Infertility, University
of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School, New Brunswick, New Jersey
Objective: Assessment of ovarian responses to metformin treatment in obese women with polycystic ovary
syndrome (PCOS).
Design: Prospective treatment with randomization to two doses of metformin.
Setting: University teaching hospital.
Patient(s): Obese women (n
82) with PCOS.
Intervention(s): Markers of ovarian function were assessed after 4 and 8 months.
Main Outcome Measure(s): Hormone (androgens and müllerian-inhibiting substance [MIS]) changes over time.
Result(s): There was no difference in the reproductive hormone changes between the doses of metformin, and
data were combined for further analyses. Significant responses to treatment were recorded for menstrual
frequency and androstenedione (A) (reduction) within the first 4 months of treatment. However, suppression of
the elevated circulating MIS concentrations required protracted treatment, because no change was observed in the
first 4 months—only in the second 4-month assessment period.
Conclusion(s): Metformin treatment of PCOS leads to rapid suppression of A and improved menstrual frequency.
Suppression of MIS is a delayed response that may be secondary to the development of a cohort of follicles that
underwent initial recruitment in an environment of reduced insulin stimulation. (Fertil Steril
2005;83:130–6. ©
2005 by American Society for Reproductive Medicine.)
Key Words: Müllerian-inhibiting substance, PCOS, metformin, ovarian follicles

Metformin has direct effects on human ovarian steroidogenesis.
Mansfield R, Galea R, Brincat M, Hole D, Mason H Fertil Steril 2003; 79:956-62.
Abstract OBJECTIVE: To investigate the possibility of direct effects of metformin on ovarian steroidogenesis. DESIGN: Cultured ovarian cells. SETTING: Academic research environment. PATIENT(S): Women undergoing bilateral salpingoophorectomy for benign gynecological disease. MAIN OUTCOME MEASURE(S): Estradiol and P were measured in granulosa cell (GC) conditioned medium and androstenedione (A) and P in theca conditioned medium. RESULT(S): The effect of addition of metformin alone to GCs was variable, but significant inhibition of both P and E2 was seen (range 0%-30%). Metformin dose-dependently inhibited gonadotrophin and insulin-stimulated P and E2 production (range 25%-50%). In theca, metformin inhibited A production (0%-40%) with no effect on P. In the presence of insulin, A was inhibited dose-dependently and P increased by a similar magnitude. CONCLUSION(S): These results demonstrate a direct effect of metformin on ovarian steroidogenesis. The inhibitory effects on androgen production in particular would be beneficial in polycystic ovary syndrome (PCOS).
MeSH Adult; Androstenedione; Estradiol; Female; Granulosa Cells; Humans; Hypoglycemic Agents; Metformin; Middle Aged; Ovarian Follicle; Progesterone; Theca Cells

Metformin alters insulin signaling and viability of
human granulosa cells
Barbara Sonntag, M.D., Martin Götte, Ph.D., Pia Wülfing, M.D., Andreas N. Schüring, M.D.,
Ludwig Kiesel, M.D., and Robert R. Greb, M.D.
Department of Obstetrics and Gynecology, University Hospital of Münster, Münster, Germany
Objective: To study whether insulin signaling pathways in the ovary are altered by metformin.
Design: In vitro human granulosa cell culture system.
Setting: Academic research environment.
Patient(s): Infertility patients undergoing oocyte retrieval for IVF/ICSI.
Main Outcome Measure(s): Cell viability and phosphorylated protein kinase B (PKB/AKT) and p44/42 mitogenactivated
protein kinase (MAPK) expression of human primary and HGL5 granulosa cells.
Result(s): Basal cell viability of primary granulosa cells was significantly increased relative to control by metformin
preincubation, without an additional stimulatory effect of insulin or IGF. Phosphorylated AKT expression in lysates of
the human granulosa cell line HGL5 was significantly increased in contrast to decreased phosphorylated MAPK
expression by metformin preincubation.
Conclusion(s): Besides systemic effects, the ovulation inducing action of metformin may at least partially be due
to direct effects on insulin signaling intermediates and follicular growth patterns in the ovary. (Fertil Steril
2005;
84(Suppl 2):1173–9. ©2005 by American Society for Reproductive Medicine.)
Key Words: AKT, granulosa, insulin signaling, MAPK, metformin, polycystic ovary syndrome

GENE EXPRESSION OF IGF-1 RECEPTOR IN HUMAN LUTEINIZED
GRANULOSA CUMULUS CELLS FROM NON OBESE
AND NON INSULIN RESISTANT WOMEN WITH POLYCYSTIC
OVARY SYNDROME (PCOS) WITH AND WITHOUT METFORMIN
TREATMENT. L. F. Santana, J. M. Ferna´ndez-Santoss, M. J. Herna´ez,
C. Caligara, R. M. Reis, M. Ferna´ndez-Sa´nchez. Department of Obstetrics
& Gynaecology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o
Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil; Department of Normal and Pathologic
Cytology and Histology, The University of Seville School of Medicine,
Sevilla, Spain; IVF Laboratory, IVI Sevilla, Sevilla, Spain; Reproductive
Medicine, IVI Sevilla, Sevilla, Spain.
OBJECTIVE: The aim of this study was to evaluate gene expression of
IGF-1 receptor in human luteinized granulosa cumulus cells from non obese
and non insulin resistant (IR) women with PCOS with and without metformin
treatment.
DESIGN: Randomized controlled clinical trial.
MATERIALS AND METHODS: We evaluated twelve women with ovulatory
cycles; 9 women with PCOS and 8 women with PCOS treated with
metformin for at least 8 weeks at a dose of 1700 mg/day. All groups were similar
regarding weight, body mass index and waist circumference. None of
them had IR. All women underwent controlled ovarian hyperstimulation using
a GnRH analogue long protocol and a personalized dose of FSHrþhMG.
Granulosa cells were obtained from the cumulus oocytes complex by microdissection
from the five largest pre-ovulatory follicles. IGF-1 receptor gene
expression was determined by semi-quantitative RT-PCR. Serum and follicular
fluid levels of E2, progesterone, testosterone, FSH, LH, insulin, SHBG
and IGF-1 were determined in each patient. For statistical analysis, ANOVA,
Newman-Keuls and Pearson’s correlation tests were used.
RESULTS: A tendency to higher expression of IGF-1 receptor gene was
observed in PCOS women without metformin treatment (33.88) . A similar
expression was detected in PCOS women who received metformin treatment
(27.5) as in the control group (24.0). The number of oocytes (20.4 vs. 13.1 vs.
11.5), the serum levels of E2 (1,896 pg/mL vs. 985.20 pg/mL vs. 908.1 pg/
mL) and testosterone (1.43 ng/mL vs. 0.89 ng/mL vs. 0.82 ng/mL) were
higher in PCOS women without metformin treatment in comparison to the
women with ovulatory cycles and PCOS women who received metformin
treatment, respectively.
CONCLUSIONS: The tendency to higher gene expression of IGF-1 receptor
observed in this study in women with PCOS without metformin treatment,
in association with higher serum levels of testosterone and E2, higher number
of oocytes retrieved, lead us to conclude that women with PCOS possibly
have higher stimulation of ovarian steroidogenesis compared to those without
this disease. The similarity of the results in this study between the
PCOS women treated with metformin and those with ovulatory cycles lead
us to hypothesize that one of the possible mechanisms of metformin action
in the IGF-1 system on the granulosa cumulus cells could be through postreceptor
mechanisms.
Supported by: This work was supported in part by a scholarship from Fundac
¸a˜o, Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior
(CAPES) - Brazil to Laura Ferreira Santana.

Hum. Reprod. Advance Access originally published online on October 14, 2008
Human Reproduction 2009 24(1):14-16; doi:10.1093/humrep/den352

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OPINION

Stringent regulation of oocyte donation in China

Boon Chin Heng¹

Ivymed International Pty Ltd (China Branch), 568 Fangxie Road, 200 011 Shanghai, People’s Republic of China

¹ Correspondence address. Tel: +86-63455050; Fax: +86-63455090; E-mail: boonchinheng@gmail.com

	Abstract

Top Abstract Funding References

 
Currently in China, health regulations permit oocyte donation only from IVF/ICSI patients who have 20 or more mature oocytes retrieved from a single cycle, of which at least 15 must be kept for their own treatment. Oocyte donation from non-patients and commercial transaction of human gametes are strictly prohibited by law. Additionally, embryos derived from donated oocytes must be cryopreserved and cannot be transferred to prospective recipients, until donors have been screened to be free of communicable diseases after 6 months. Such overly stringent regulation has in turn led to a severe shortage of available donor oocytes in China. The situation is made worse by a cultural aversion to oocyte donation by the majority of patients, because biological kinship and blood relations are viewed as sacrosanct in traditional Chinese culture. The harsh social stigmatization of childlessness in Chinese society, increasing incidence of age-related female infertility in recent years and growing numbers of bereaved older women who have lost their only child to accidents, natural disasters and suicides would make it imperative to reconsider liberalizing the regulation of oocyte donation in China. In particular, the blanket ban on oocyte donation by non-patients should be lifted, as it is anticipated that there are many young healthy women in China who are generous and open-minded enough to consider altruistically donating their oocytes to childless couples.

Key words: donation/ethics/oocyte/recipients

In 2006, the Health Ministry of China promulgated a new set of legislation pertaining to oocyte donation (Han, 2006). According to Decree No. 44 of 2006 (Health Ministry of China, 2006), donor oocytes can only be procured from IVF/ICSI patients, and solicitation of oocyte donation from non-patients is explicitly forbidden. Furthermore, oocyte donation is permitted only if 20 or more mature oocytes are retrieved from the patient, with at least 15 oocytes being retained for the patient’s own use and the remainder can be donated. Fresh embryos produced from donor oocytes must be cryopreserved and cannot be transferred in a fresh cycle. Six months later, oocyte donors must then be retested for the human immunodeficiency virus, hepatitis B virus, hepatitis C virus and other relevant communicable diseases. Only if the oocyte donor is tested and found to be negative for these communicable diseases, the cryopreserved embryos obtained from donor oocytes can be thawed and transferred to the prospective recipient. Any commercial transaction involving donor oocytes is prohibited, and approval must be obtained from the local provincial or municipal health authority before oocyte donation service is provided.

As expected, such overly stringent regulation has led to a severe shortage of available donor oocytes and consequently long waiting lists of prospective recipients at virtually all fertility clinics within China. It is rather uncommon to find IVF/ICSI patients with 20 or more retrieved mature oocytes. Even then, not all such patients are willing to share their excess oocytes with another patient, particularly if they are childless and attempting to conceive themselves. The situation is made worse by a cultural aversion to oocyte donation by the majority of Chinese patients. In traditional Chinese culture, biological kinship and blood relations are viewed as sacred, and it would be an insult to one’s family and ancestors, as well as a personal tragedy to have unknown and unacknowledged biological descendants. It must be noted that Confucian and Taoist tradition place much emphasis on the ‘spiritual connection’ between ancestors and their biological descendants, through the rite of ancestor worship that is currently practiced by a large segment of the Chinese population.

Although there is always some social stigma associated with infertility, gamete donation and adoption in almost every human culture, stigmatization of childlessness is particularly harsh in Chinese society: it is seen both as a curse and as a major failing in filial duty to one’s parents and ancestors (Qiu, 2002) because the Confucian rite of ancestor worship would require living biological descendants to carry out prayer offerings for the deceased. It is not uncommon for childless couples to face embarrassing questions from close relatives and friends at social gatherings during major Chinese festivals, such as the Lunar New Year.

There is a much stronger aversion against child adoption in Chinese culture, when compared with Western societies where it is a more common and acceptable option for childless couples. This is because of the patriarchal nature of Chinese society, in which male bloodlines and clan ancestry are paramount to an individual’s identity (Qiu, 2002). No doubt, oocyte donation entails a loss of genetic connectedness to the birth mother with its inherent psycho-social implications on the parent–child relationship (van den Akker, 2006; Purewal and van den Akker, 2007). Nevertheless, what really matters in the traditional Chinese family value system is that biological kinship to the father and patrilineal clan ancestry is preserved. Oocyte donation is thus very much preferable to the adoption of children from unrelated families in the Chinese cultural perspective. In fact, Confucian tradition sanctions adoption only among close relatives belonging to the same patrilineal clan or extended family, and sharing the same surname. However, rigorous implementation of the one-child policy (Mosher, 2006) in China over the past few decades has made it virtually impossible for childless couples to adopt from their close relatives.

Chinese patients requiring oocyte donation fall into four distinct categories: (i) women without functional ovaries, i.e. Turner’s syndrome (Foudila et al., 1999) or afflicted with premature ovarian failure (Kalantaridou et al., 1998); (ii) women with poor quality oocytes and recurrent IVF/ICSI failures, i.e. polycystic ovary syndrome (PCOS, Patel and Carr, 2008); (iii) older women who had either married late in life or deliberately delayed childbearing after marriage (Friese et al., 2006); (iv) bereaved older mothers who had lost their children to traffic accidents, suicides and natural disasters. The first category of prospective recipients without functional ovaries or afflicted with premature ovarian failure is relatively rare in the Chinese population, and the second category is primarily composed of PCOS patients who constitute 2–3% of Chinese women of reproductive age (Chen et al., 2008). The third category, composed mostly of women attempting to have children in their late 30s or early 40s (Friese et al., 2006), is becoming more common due to the increasing trend of highly educated urban Chinese women deliberately choosing to delay marriage and childbearing in pursuit of career and educational goals. This trend is further exacerbated by sky-rocketing property prices in large Chinese cities, which has prompted many newly wed couples to delay having children until they can stabilize their financial and housing situation. The fourth category, composed of bereaved older mothers is also becoming more common, due to China’s one-child policy (Mosher, 2006), and the rising numbers of traffic-related deaths and suicides of young people in China. The escalating number of motor vehicles spurred by rapid economic growth and resulting congestion of China’s roads and highways has dramatically increased the incidence of traffic-related deaths of young people in recent years (Yan-Hong et al., 2006; Wang et al., 2008). Additionally, suicide of young adults and children in their late teens is also becoming more prevalent (Liu and Tein, 2005; Jiang et al., 2007), due in large part to a highly competitive college entrance examination system (GaoKao), which allows only an extremely small proportion of candidates to enter reputable colleges and universities (Siegel, 2007). The bulk of successful candidates (50–60%) entering higher education often have to contend with poorly funded sub-standard colleges and universities that provide bleak job prospects upon graduation (Plafker, 2004). As a result, many Chinese children are pushed extremely hard by their parents to excel academically at a young age, and their entire life often revolves around studies and examination. High parental expectation is further exacerbated by China’s one child policy (Mosher, 2006), resulting in many parents and even grandparents pinning all their hopes and aspirations on only one child. Hence, it is not uncommon for many young people to feel a strong loss of self-esteem and meaning in life, if they were to fail academically, and subsequently contemplate suicide (Liu and Tein, 2005; Jiang et al., 2007).

More recently, the Sichuan earthquake (May 2008) in south-west China, which led to the death of some 4700 school children (Branigan, 2008), brings home the point on much needed reforms in the regulation of oocyte donation. Many bereaved mothers who lost their only child in the earthquake are in their late 30s and early 40s and consequently have difficulties in conceiving another child. Although the Chinese central government has made an extraordinary effort in providing disaster relief and reconstruction of the affected areas, the lost children can never be brought back to life. Under such circumstances, the liberalization of oocyte donation would not only give hope to many bereaved older mothers in conceiving another child, but also provide an opportunity for young healthy women to help those bereaved mothers through oocyte donation.

There is also a risk that the requirement for prospective donors to have 20 or more retrieved mature oocytes may encourage the injudicious use of unnecessarily high gonadotrophin dosages on good prognosis patients (Heng, 2007). To maximize the number of retrievable oocytes, prospective donors will often be restricted to younger women with mild fertility problems (i.e. fallopian tube occlusion) or with male partner infertility (Heng, 2007, 2008), because such patients readily produce many oocytes under gonadotrophin stimulation. There has been recent scientific evidence that such good prognosis patients would do better either without exogenous gonadotrophins (natural cycle) or with minimal ovarian stimulation (Edwards, 2007; Ubaldi et al., 2007). In their case, although fewer mature oocytes are retrieved, the quality is better (Fauser et al., 1999), and there is also improved endometrial receptivity and luteal support for embryo implantation (Devroey et al., 2004; Lindhard et al., 2006). Additionally, high gonadotrophin dosages are associated with increased risks of ovarian hyperstimulation syndrome (Budev et al., 2005) in ‘good-responder’ patients. It would thus be unethical to subject good prognosis younger women with mild fertility problems or male partner infertility to high hormone dosages, just for the sake of maximizing the number of retrievable oocytes available for donation. Also, it must be remembered that recombinant gonadotrophins used in fertility treatment are expensive and form a substantial portion of the medical fees (Gleicher et al., 2003).

The tremendous shortfall of donor oocytes in China, coupled with the strong social stigma suffered by many childless couples, would make it imperative to re-examine the overly stringent regulation of oocyte donation, particularly the requirement for patients to have 20 or more retrieved mature oocytes in order to qualify as donors. Additionally, the blanket ban on oocyte donation by non-patients should also be reconsidered. It is anticipated that there are many young healthy women in China who are generous and open-minded enough to consider altruistically donating their oocytes to aid in the conception of childless couples, if permitted to do so. In fact, there are more ethical challenges in soliciting oocyte donation from IVF/ICSI patients since they are attempting to conceive themselves and it would be a daunting prospect for them to fail at clinical assisted reproduction, while being unsure of whether their donation has resulted in a successful conception by another woman (Heng, 2008). The plight of bereaved older mothers who have lost their only child needs special consideration. Surely, these women deserve an opportunity to conceive another child through oocyte donation.

	Funding

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Funding for this study was provided by Ivymed International Pty Ltd (China Branch).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Funding References

 
Donors
All donated oocytes came from the Reproductive Medical Center of the First Affiliated Hospital of SUN YAT-SEN University, which is certified by the Ministry of Health of the People’s Republic of China. All oocyte donors were on an ART cycle. No financial benefit was involved in the donation process. Oocyte donors were clearly informed of all the study details, including the oocyte’s use and research destination, and they voluntarily signed detailed informed consent documents. We guaranteed that all oocytes would be used in basic scientific research and not for reproductive purposes.

While conducting ART, we collected oocyte donations under the therapeutic cloning guidelines passed by the Ministry of Health of the People’s Republic of China. We considered donation as an option when >30 oocytes were collected from a patient. Normally, only one to four oocytes were obtained from a donor and they were selected stochastically, so as not to have any impact on the patient’s clinical treatment.

Ovulation induction and oocyte retrieval
A total of 21 couples were involved in this study (17 donors for SCNT and four donors for parthenogenetic activation). Each patient underwent a basic physical examination before ovulation induction, including tests for human immunodeficiency virus, hepatitis B virus, hepatitis C virus and contagious venereal disease. Patients had regular menstrual cycles every 29–32 days. Starting in the luteal phase of the previous cycle, on cycle day 21, eligible patients received 1.3 mg of a gonadotrophin-releasing hormone agonist (Dapherin) and then recombinant follicle-stimulating hormone (Gonal-f, Serono, Sweden) at a dose of 150–300 IU/day from Day 3 of the menstrual cycle to promote the growth of multiple follicles. When the diameter of dominant follicles reached 18 mm, a single dose of 10 000 IU of human chorionic gonadotrophin (Profasi, Serono, Sweden) was administered and transvaginal follicular aspiration was performed 36 h later. The cumulus–oocyte complex was cultured in culture medium (Quinn’s Advantage^TM Fertilization Medium with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute, SAGE IVF, Inc.) for 3–4 h in at 37°C in a humidified atmosphere of 5% CO₂, 5% O₂ and 90% N₂ before SCNT.

Oocyte classification by morphologic criterion
Maturation and morphological features of the oocytes were investigated immediately before SCNT manipulation. Analyzed anomalies included dark central granulation of the cytoplasm, refractile bodies, vacuoles, aggregation of smooth endoplasmic reticulum (sER) an abnormal zona pellucida and an irregular first polar body (De Sutter et al., 1996; Ebner et al., 2003).

A normal matured MII oocyte should have a clear, moderately granulate cytoplasm, a small perivitelline space and an intact first polar body. Oocyte morphology criterion should be divided into two parts: one assesses extracytoplasmic abnormalities, including the shape of the oocyte itself, enlargement of the perivitelline space, presence of debris in the space and fragmentation of the first polar body, while the other determines if the cytoplasm is abnormal, with granulation and aggregation of the sER. Oocytes in the first grade or group are round with a smooth first polar body, dispersed cytoplasmic granula and normal perivitelline space (the widest space is similar to the diameter of the first polar body). The oocytes in the second group have similar morphology except slightly centralized granula. The oocytes in the third group are totally different from the first two groups. Although the oocyte morphology is round, the first polar body is inconspicuous, the morphology is fragmented or degenerated, the granula status is extensive centralization, and importantly the widest part of the perivitelline space is much smaller than the diameter of a normal first polar body. Sometimes there is no space which resembles an MI oocyte. The oocytes in the forth group have abnormal morphology, dispersed granules, and the widest part of the perivitelline space is much bigger than the diameter of a normal smooth first polar body with the oocytes able to wander in the perivitelline space; however, the morphology of the first polar body is similar to that in the third group (Fig. 1).

View larger version (138K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1 Four types of oocytes used in human SCNT experiments. (A) Grade A oocytes could support donor nuclei to develop into a blastocyst with high efficiency. (B) Grade B oocytes could support donor nuclei to develop into a blastocyst, but with low efficiency. (C) Grade C oocytes could not support donor nuclei to develop beyond the morula stage, but could be fertilized by IVF and could then develop further. (D) Grade D oocytes could support neither donor nuclei nor sperm.

 
Preparation of donor cells
Donor cells were obtained by foreskin excision during the surgical procedure on a 3-year-old boy, and were named HFSF-1. The cells were cultured in medium containing 10% FBS and 90% low-glucose DMEM (Gibco) and were cryopreserved after two passages. At 48 h before NT, the fibroblasts were thawed and cultured for 2 days. Single cells were retrieved by trypsinization, and only cells in Passages 4–6 were used as donor cells (Fig. 2).

View larger version (123K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2 Morphology of HFSF-1 donor cells (x200 under an inverted microscope).

 
Karyotype and cell cycle of donor cells
Foreskin fibroblasts were incubated for 3 days and then collected and subjected to trypsinization. After washing with phosphate-buffered saline (PBS) and incubation in 0.075 mol/l potassium chloride for 10 min at 37°C, cells were fixed three times with 1:3 methanol/glacial acetic acid and dropped onto glass slides. Chromosome spreads were Giemsa-banded and photographed (Fig. 3). The karyotype of donor cells was determined in Passage 4–6, the same time as the cells were used in SCNT experiments.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3 Karyotype of HFSF-1 donor cells at Passage 8.

 
HFSF-1 cells were cultured to 80% confluence and then washed in PBS without Ca²⁺ or Mg²⁺. After treatment with 0.5% trypsin, the cells were collected and centrifuged at 350 g for 5 min at 4°C. The cell pellet was collected and the centrifugation was repeated. The cell pellet (1.5 x 10⁶ cells) was then resuspended in 70% ice-cold alcohol and stored at 4°C overnight. The next day the cells were centrifuged at 350 g for 10 min at 4°C and then resuspended in ice-cold PBS. After washing twice in ice-cold PBS, the cells were centrifuged again at 350 g for 10 min at 4°C. Then, 500 µl of propidium iodide/Triton X-100 staining solution was used to resuspend the cells, and 100 µl/ml DNAse-free RNAse A was added. After incubation for 20 min at 37°C, the sample was stored at 4°C and protected from light. The cells were analyzed by flow cytometry within 48 h, and the data were assessed using MulticycleAV (IBM-PC) (Fig. 4).

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4 Cell cycle distribution of HFSF-1 donor cells.

 
Somatic cell nuclear transfer
Cumulus–oocyte complexes were cultured in vitro in culture medium (Quinn’s Advantage^TM Fertilization Medium with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute), and then treated with 80 IU/ml hyaluronidase 30 min before SCNT micromanipulation. Mechanical handling was performed after brief hyaluronidase treatment. The oocytes were then cultured in an incubator for 30 min to balance the effects of in vitro manipulation. After this, three to four oocytes were transferred into a droplet of HEPES medium (Quinn’s Advantage^TM Medium with HEPEs with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute) containing 5 µg/ml cytochalasin B, and placed in an operation chamber on the microscope stage. The spindle, observed using Spindle View (Cri Inc.), was fixed at the 3 o’clock position using a hold needle. The one-step method reported by Zhou et al. (2003, 2006) was used; an injection needle was used to break down the zona pellucida using a piezo current, a donor cell was selected to inject into the cytoplasm along the cut, and then the spindle oocyte was removed.

After manipulation, reconstructed embryos were immediately transferred back into culture medium (Quinn’s Advantage^TM Fertilization Medium with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute) at 37.5°C and incubated for 2 h before activation. The reconstructed embryos were to be divided, according to oocyte morphology criterion, into different drops for culture.

Embryo activation and culture
According to our previous report (Mai et al., 2007), electric-activation was combined with chemical treatment to activate the reconstructed embryos. Electric-activation was performed in 0.3 M mannitol medium without calcium, and involved stimulation with two 20-µs pulses of 1.6 kV/cm using an electro cell manipulator (BTX 2001, San Diego, CA, USA). Reconstructed embryos were rinsed and exposed to 5 µM ionomycin (Sigma–Aldrich) for 5 min. After extensive washing in HEPES-buffered medium, the embryos were incubated at 37°C under 5% CO₂ in humidified air at 1.9 or 2 mM 6-DMAP (Sigma–Aldrich) for an additional 5 h.

After 6-DMAP treatment, groups of two to three reconstructed embryos in the same category were cultured in one droplet of cleavage culture medium (Quinn’s Advantage^TM Cleavage Medium with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute) under mineral oil (Sigma–Aldrich) at 37°C in a humidified atmosphere of 5% CO₂, 5% O₂ and 90% N₂. After 68–72 h of embryo activation, the embryos that developed to the 8-cell stage were transferred to blastocyst culture medium (Quinn’s Advantage^TM Blastocyst Medium with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute) for sequential culture to the blastocyst stage.

Parthenogenetic activation
While performing the SCNT procedure, some fresh MII oocytes were retained for parthenogentic activation. Immature oocytes identified during the treatment of cumulus–oocyte complexes were incubated for a further 24 h (Quinn’s Advantage^TM Cleavage Medium with 12% v/v Quinn’s Advantage^TM SPS Serum Protein Substitute). Oocytes that exhibited a first polar body were considered mature and were also subjected to parthenogenetic activation. Activation and culture conditions were the same as for SCNT embryos.

Statistical analysis
All experiments, including SCNT experiment, karyotyping indentification and flow cytometry, were repeated at least three times. Embryo developmental data were analyzed by t-test using SPSS 13.0 software. Statistical significance was accepted at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Funding References

 
Karyotype and cell cycle of HFSF-1 donor cells
HFSF-1 cells retained the normal morphology of fibroblasts during propagation (Fig. 2) and exhibited the 100% normal diploid 22-XY karyotype (n= 50) in Passage 4–6, which is when they were used in SCNT experiments (Fig. 3).

Different phases of the cell cycle in HFSF-1 cells were detected by flow cytometry (Fig. 4). The intense DNA peaks (represented by the blue grids) show that the majority of HFSH-1 cells at 80% confluence were in G0/G1-phase (60.5–65.6%). The distribution of S-phase (represented by light red diagonals) was 20.8–30.8%, and that of G2/M-phase was 8.7–11.6%.

Assessment of developmental efficiency
To investigate the development potential of human oocytes after artificial activation, some oocytes were subjected to parthenogenetic activation without an SCNT attempt, and the developmental efficiency was observed and recorded every 24 h. From a total of eight oocytes, seven fresh oocytes were activated after culture for 2 h in an incubator; of these, all embryos developed to the 2-cell stage, and two successfully progressed to blastocysts (Table I). Such blastocysts are capable of expanding and hatching, and these indeed hatched as normally fertilized blastocysts (Fig. 5I). In addition, a total of 28 GV or GVB oocytes were identified during cumulus–oocyte complex treatment, and 18 of these spontaneously expelled the first polar body after a further 24 h in in vitro culture. After artificial activation treatment, there was no difference from the cleavage rate between fresh and IVM oocytes; however, the activation rate and the blastocyst-development efficiency of fresh oocytes were significantly higher than for IVM oocytes (Table I).

View this table: [in this window] [in a new window]   Table I Development efficiency of parthenogenetic embryos

 

View larger version (125K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5 SCNT embryo development process in vitro. (A) Donor cells from foreskin fibroblast, (B) fresh MII oocyte, (C) pseudo-pronuclear embryo, (D) 2-cell stage, (E) 4-cell stage, (F) 8-cell stage, (G) expanded blastocyst and (H) hatching blastocyst. (I) Expanded blastocyst after parthenogenetic activation.

 
Oocyte morphology score for prediction of embryo development
Poor-quality and immature oocytes have been regarded as a viable resource for human SCNT from an ethical point of view, but actual manipulation usually results in failure. To investigate this issue, we classified all donated oocytes into four categories using morphology criteria in our ART center (Table II).

View this table: [in this window] [in a new window]   Table II Morphological classification of oocytes

 
Overall, regardless of oocyte group, 91.8% of the 61 oocytes survived after piezo-activation; of these surviving oocytes, 69.6% were activated with 39.3% cleaving to at least the 2-cell stage (Table III). Embryos from oocytes of Grades A and B developed with similar efficiency (33.3 versus 25%, respectively). However, embryos from Grade C oocytes developed no further then the 8-cell stage before arresting. Embryos from Grade D oocytes were activated with low efficiency and none could develop to the 2-cell stage (Table III). Good morphology and a distinct inner cell mass could be observed in the cloned blastocysts which had the ability to hatch (Fig. 5A–H).

View this table: [in this window] [in a new window]   Table III Effect of oocyte morphology on the development efficiency of human SCNT embryos

 

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Funding References

 
Human blastocysts can be obtained using SCNT technology
In the present study, human cloned blastocysts were obtained using a piezo-assisted injection method, which is different from the traditional electrofusion method previously described (Stojkovic et al., 2005). Oocytes are regarded as a valuable resource because of their limited numbers and the ethical issues involved, and the reconstruction efficiency using electrofusion is very low compared with the piezo-assisted injection method (Yu et al., 2007a, b). Comparison of these NT methods has been reported in some species, including the pig (Kurome et al., 2003), mouse (Yu et al., 2007a, b) and rhesus monkey (Zhou et al., 2006). Our previous mouse SCNT experiments suggested that there is no difference in blastocyst formation between the two SCNT methods, but the reconstruction efficiency is different. It is apparent that more oocytes are used for the electrofusion method, although a recent study reported increased fusion efficiency (Li et al., 2005). Compared with a recent human SCNT study (Stojkovic et al., 2005), our higher efficiency of activation and cleavage and similar efficiency of blastocyst formation for oocytes of Grade A or B was mainly attributed to the NT technology itself. Our previous study indicated that micronuclei are induced by piezo-assisted NT, and this is similar to apoptosis post-implantation (Yu et al., 2007b), but it is not harmful to blastocyst development. Thus, piezo-assisted methods are better than electrofusion techniques from the point of view of oocyte usage.

G0/G1 or pre-S-phase donor nucleus exhibita capacity for reprogramming when placed in a MII oocyte with a high level of maturation promoting factor, is shown in SCNT studies of many species. The usual methods of cell cycle synchronization of cultured cells are serum starvation and contact inhibition. Serum starvation is widely used for synchronizing somatic cells by arresting them in the G0/G1 phase of the cell cycle, but aberrant gene transcription has been observed in human primary fibroblasts treated using serum starvation method (Iyer et al., 1999). Also serum starvation method often reduces cell survival and increases DNA fragmentation (Kues et al., 2000), which causes high-embryonic losses after NT (Lawrence et al., 2005). Growing cells to confluence is another strategy used for synchronizing cells at G0/G1 stage of the cell cycle, and in the present study, 65% of the cells were at G0/G1 after culturing to confluence. Although our efficiency is lower compared with the previous report in porcine (Boquest et al., 1999), which is possibly attributed to less confluence of somatic cells because the cells were only thawed and cultured for no more than 48 h, our approach coupled with assessment of cell diameter and other criteria, was sufficient to select a suitable donor cells.

Oocytes score aids in evaluating oocyte developmental potential
Oocyte quality is regarded as a key factor in SCNT because the oocyte provides the space and materials for reprogramming of donor genetic materials, as well as the nutrition and substances required during further embryo development. The hope that IVM oocytes could be used for reprogramming using somatic cell nuclei (Takeuchi et al., 1999; Hall et al., 2007) arose from successful pregnancies established using IVM oocytes (Buckett et al., 2007). However, some researchers have reported low efficiency for IVM compared with fresh oocytes, with IVM oocytes even exhibiting abnormal expression of Oct-4, an important factor in embryo differentiation (Chen et al., 2004; Chang et al., 2005). Our parthenogenetic activation results suggest that the developmental potential of such oocytes is limited during the IVM process. IVM oocytes represent a tremendous cloning resource, but their low development potential remains a problem. Serial SCNT seems to represent a solution for cloning in mice (Heindryckx et al., 2002), but in humans it is impossible to obtain a normal zygote after a second SCNT.

Zygote scores have been predominantly used in ART, and some studies have indicated that such a score could predict the development and pregnancy efficiency of embryos in patients treated by IVF or ICSI (Edirisinghe et al., 2005; Payne et al., 2005). Although there are conflicting results from different ART centers (James et al., 2006; Nicoli et al., 2007), we still consider that a scoring system would be helpful in embryo classification and research. Oocyte classification is still scarcely reported, although zygote scores have been widely investigated. Serhal et al. analyzed ICSI results based on oocyte morphology and reported that while early and preimplantation development efficiency was not affected by oocytes of abnormal morphology or cytoplasm-containing oocytes, implantation failed for such abnormal oocytes (Ebner et al., 2003). In our SCNT study, we established oocyte grades based on maturation signs, including oocyte morphology, first polar body, cytoplasmic granula distribution and perivitelline space. Owing to different physiological reactions in patients to extrinsic hormones, different types of oocytes can be obtained from the ovulation induction procedure (Racowsky et al., 2005). Although strict criteria for oocyte collection have been established, including B-type ultrasonic inspection, follicular volume and clinical examination, so that few immature oocytes are obtained, there are still differences among mature MII oocytes.

A mature nucleus and cytoplasm are regarded as equally important for embryo development, and each is closely associated with oocyte morphology. The occurrence of the first polar body is proof of nuclear maturation, but another issue is to identify an evaluation standard for cytoplasm maturation. Moreover, cytoplasm contains accumulating materials, and provides proteins, RNA, morphorgenetic factors and protective chemicals. Thus, a mature cytoplasm seems to play a key role in reprogramming of the donor cell genome. Our results indicate that oocytes of grade A or B had the ability to support SCNT embryo development to the blastocyst stage, however when using oocytes of grade C, the reconstructed embryos arrested at or before the 8-cell stage, which indicated that the zygotic genome was incompletely activated for such a reprogramming environment.

Although different studies have shown varying ICSI results (De Sutter et al., 1996), oocyte quality is very important in SCNT. Successful SCNT is also highly dependent upon oocyte quality, as demonstrated in ferret cloning (Li et al., 2006). In a bovine study, oocytes from different backgrounds had different effects on cloned embryo development in vitro, and this differed from the effect on IVF embryos (Yang et al., 2008). According to our own experiments in mice, oocytes from mice of different backgrounds and ages exhibit different blastocyst development efficiency, even for embryonic stem cell establishment (data not shown).

Future perspectives
Although the present results are encouraging, the numbers of oocytes of Grades A, B and D were small, and the findings require verification with large sample sizes. SCNT still has low efficiency and is affected by many factors, including donor cells, recipient oocytes, NT technology and method, activation treatments, etc. Therefore, a predictable standard should be established for human SCNT because human oocytes are a rare resource. Recently, the potential of induced pluripotent stem (iPS) cell technology has been identified for therapeutic cloning (Takahashi et al., 2007; Yu et al., 2007a) and the technique was successfully applied in a mouse model (Hanna et al., 2007). However, some problems need to be resolved before clinical application, including consideration of virus utility, epigenetic analysis, etc. (Stojkovic and Phinney 2008). Therefore, it cannot be assumed that iPS will replace ES cells in clinic therapy because of their lack of moral and ethical issues. For clinical therapeutic cloning, the realistic approach is still derivation of stem cell lines from cloned human blastocysts, because more studies have been attempted in ES cells, including differentiation, self-renewal and therapeutic security (tumor formation and epigenetic stability). Here, we set up an evaluation standard that predicts the development potential of human SCNT embryos and may help in the production of human SCNT blastocysts for therapeutic cloning.

Hum. Reprod. Advance Access originally published online on December 9, 2008
Human Reproduction 2009 24(3):649-657; doi:10.1093/humrep/den407

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Assessment of the developmental competence of human somatic cell nuclear transfer embryos by oocyte morphology classification

Yang Yu^1,2,, Qingyun Mai^3,, Xinjie Chen^1,2,, Liu Wang¹, Ling Gao³, Canquan Zhou^3,4 and Qi Zhou^1,4

¹ State Key Laboratory of Reproductive Biology, Institute of Zoology, Datun Road, Chaoyang District, Beijing 100101, People's Republic of China ² Graduate School, Chinese Academy of Sciences, Beijing, People's Republic of China ³ Reproductive Medical Center, First Affiliated Hospital of SUN YAT-SEN University, Zhongshan Er Road 58, Guangzhou, Guangdong 510080, People's Republic of China

⁴ Correspondence address. Tel:/Fax: +86-10-64807299; E-mail: qzhou@ioz.ac.cn (Q.Z.), Tel:/Fax: +86-20-87786029; E-mail: zhoucanquan@hotmail.com (C.Z.)

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Funding References

 
BACKGROUND: The oocyte plays a key role in reprogramming the epigenetic status of donor cell nuclei, and the absence of reprogramming elements in the cytoplasm or aberrant accumulation of proteins can trigger the abnormal development of nuclear transfer (NT) embryos. Previous studies have demonstrated the relationship between oocyte morphology and both embryo development and pregnancy outcome. In the present study, we compared the morphology of oocytes with subsequent development of human somatic cell NT (SCNT) embryos.

METHODS: Piezo-assisted SCNT technology was used to produce reconstructed embryos, with almost 92% of oocytes reconstructed successfully. Depending on their morphologies, we separated metaphase II oocytes into four grades according to criteria which assess oocyte morphology, first polar body and perivitelline space, and especially, cytoplasm granula distribution.

RESULTS: Embryos from oocytes of Grades A and B could develop to the blastocyst stage with similar development efficiency for every developmental stage. However, embryos from Grade C oocytes arrested at or before the 8-cell stage then degraded, and the donor cell genome could not be activated and reprogrammed in such oocytes. For Grade D oocytes, cleavage was not observed in the reconstructed embryos, suggesting that the oocytes themselves have no developmental potential.

CONCLUSIONS: Our study revealed that different levels of developmental competence of SCNT embryos resulting from different oocyte reprogramming potentials associated with different morphologies. The results suggest that effective methods for improving oocyte quality should be studied, and that human SCNT efficiency would be increased following simple assessment of established oocyte morphology criterion.

Key words: human somatic cell nuclear transfer (SCNT)/oocyte morphology/oocyte donation/blastocyst development

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Funding References

 
Stem cell therapy has tremendous treatment potential for replacing certain damaged cells, with proven feasibility in the mouse (Rideout et al., 2002; Barberi et al., 2003). However, the problem of immune incompatibility remains unresolved. Establishment of a homologous embryonic stem cell line derived from patient autologous cells using somatic cell nuclear transfer (SCNT) technology is widely regarded as a feasible solution, with success in the mouse and a non-human primate (Wakayama et al., 2001; Byrne et al., 2007). However, there are obstacles in the production of human blastocysts using SCNT technology, although one successful human SCNT study was recently reported (Stojkovic et al., 2005; French et al., 2008).

Interspecies studies have indicated that bovine and rabbit oocytes are capable of reprogramming human somatic cells, and interspecies blastocysts were produced (Chen et al., 2003; Chang et al., 2004). However, owing to ethical issues, such interspecies embryos can only be used in scientific research and are a long way from clinical application (Tecirlioglu et al., 2006; Minger, 2007). Recently, scientists in different laboratories have performed human SCNT experiments and tried to derive human SCNT blastocysts; these developed to the morula stage, but failed to develop to the blastocyst stage (Hall et al., 2007; Heindryckx et al., 2007). Successful derivation of human SCNT blastocysts was reported in 2005 (Stojkovic et al., 2005), and recently French et al. (2008) reported that human SCNT blsastocysts were successfully derived with higher efficiency from adult fibroblasts.

With the development of medical technology to assess oocytes prior to retrieval, follicular volume can be observed using B-type ultrasonic inspection and the oocytes in the follicles with determinate diameters can be regarded as matured in vivo. However, metaphase II (MII) oocytes collected from follicles are not uniform because of individual differences resulting from ovarian stimulation and the hormonal environment (Racowsky et al., 2005). In clinical treatment, some studies have reported successful pregnancy after embryo transfer using low-quality MII oocytes (Serhal et al., 1997; Balaban et al., 1998), but with lower efficiency compared with normal MII oocytes. For SCNT, the cytoplasm of the oocyte is regarded as the key environment for reprogramming the epigenetic status of donor cell nuclei. The molecular mechanisms of oocyte reprogramming remain unclear; however, poor-quality oocytes are probably deficient in reprogramming elements in the cytoplasm or have aberrant accumulation of proteins, such as major vault protein, which triggers the abnormal development of nuclear transfer (NT) embryos, even in fertilized embryos (Gioia et al., 2005; Sutovsky et al., 2005). It is impossible to perform molecular and biochemical testing of oocyte quality before SCNT. Therefore, it is necessary to establish a simple criterion to predict the oocyte’s ability to reprogram the donor nucleus and to allow the subsequent development of an SCNT embryo.

To investigate this issue, we separated donated oocytes into four categories based on clinical standards in our assisted reproductive technology (ART) center, and compared their development efficiency after SCNT. Our aim was to establish an evaluation standard to predict the development potential of human SCNT embryos and to aid in understanding the normal mechanism of cytoplasmic reprogramming of human nuclei.

Hum. Reprod. Advance Access originally published online on December 4, 2008
Human Reproduction 2009 24(3):496-501; doi:10.1093/humrep/den398

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OPINION

Management of endometriomas in women requiring IVF: to touch or not to touch

Juan A. Garcia-Velasco^1,* and Edgardo Somigliana²

¹ IVI Madrid, Rey Juan Carlos University, Av. del Talgo 68, Madrid 28023, Spain ² Infertility Unit, Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena, Milan, Italy

^* Correspondence address. E-mail: jgvelasco@ivi.es

	Abstract

Top Abstract Introduction Endometriomas and ovarian... Endometrioma-related impact on... Treatment prior to IVF The risks of surgery... Conclusions and recommendations Acknowledgements References

 
The classic, unproven dogma that ovarian endometrioma should be removed in all infertile women prior to IVF has been recently questioned. There is currently insufficient data to clarify whether the endometrioma-related damage to ovarian responsiveness precedes or follows surgery. Both endometrioma-related injury and surgery-mediated damage may be claimed to be involved and the relative importance of these two insults remains to be clarified. Convincing evidence has emerged showing that responsiveness to gonadotrophins after ovarian cystectomy is reduced. Conversely, the impact of surgery on pregnancy rates is unclear since no deleterious effect has been reported. Of relevance here is that surgery exposes women to risk related to a demanding procedure whereas risks associated with expectant management are mostly anecdotal or of doubtful clinical relevance. We recommend proceeding directly to IVF to reduce time to pregnancy, to avoid potential surgical complications and to limit patient costs. Surgery should be envisaged only in presence of large cysts (balancing the threshold to operate with the cyst location within the ovary), or to treat concomitant pain symptoms which are refractory to medical treatments, or when malignancy cannot reliably be ruled out.

Key words: ovarian endometriomas/surgery/IVF/ovarian responsiveness/pregnancy rates

Hum. Reprod. Advance Access originally published online on February 6, 2009
Human Reproduction 2009 24(5):1126-1132; doi:10.1093/humrep/dep015

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DNA methyltransferase expression in the human endometrium: down-regulation by progesterone and estrogen

Yoshiaki Yamagata, Hiromi Asada, Isao Tamura, Lifa Lee, Ryo Maekawa, Ken Taniguchi, Toshiaki Taketani, Aki Matsuoka, Hiroshi Tamura and Norihiro Sugino¹

Department of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan

¹ Correspondence address. Tel: +81-836-22-2286; Fax: +81 836-22-2287; E-mail: sugino@yamaguchi-u.ac.jp

BACKGROUND: Epigenetic regulation may be involved in modulation of gene expression during the normal cyclic changes of the human endometrium. We investigated expression of DNA methyltransferases (DNMTs) in endometrium during the menstrual cycle and the influence of sex steroid hormones on DNMT in endometrial stromal cells (ESC) in culture.

METHODS: Expression of DNMT1, DNMT3a and DNMT3b was assessed by immunohistochemistry and real-time RT–PCR in endometrial tissue (n = 42 women). ESC (n = 3 women) were cultured with estradiol and medroxyprogesterone acetate (E + MPA) for 17 days, and DNMT mRNA levels were measured by real-time RT–PCR.

RESULTS: Nuclei of both epithelial and stromal cells immunostained for DNMT1, DNMT3a and DNMT3b during each phase of the menstrual cycle. Tissue levels of DNMT1 and DNMT3a mRNA were significantly lower in the mid-secretory phase than in the proliferative phase (P < 0.01). For DNMT3b, the change in mRNA levels showed a similar trend to that for DNMT3a. In ESC culture, DNMT3a and DNMT3b mRNA levels were significantly decreased by E + MPA treatment (P < 0.01 and P < 0.05, respectively) at Day 8 and Day 17.

CONCLUSIONS: DNMT mRNAs declined in the human endometrium during the secretory phase, and E + MPA down-regulated DNMT3a and DNMT3b mRNAs in ESC in culture. These results suggest that DNMTs have regulatory functions in gene expression that is associated with decidualization.

Key words: DNA methyltransferase/endometrium/endometrial stromal cell/decidualization

Hum. Reprod. Advance Access originally published online on February 13, 2009
Human Reproduction 2009 24(5):1051-1058; doi:10.1093/humrep/dep018

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Single Day 2 embryo versus blastocyst-stage transfer: a prospective study integrating fresh and frozen embryo transfers

F. Guerif^1,2,3, M. Lemseffer^1,2,3, R. Bidault^1,2,3, O. Gasnier^1,2,3, M.H. Saussereau^1,2,3, V. Cadoret^1,2,3, C. Jamet^1,2,3 and D. Royere^1,2,3,4

¹ Service de Médecine et Biologie de la Reproduction, CHRU Bretonneau, 2 Boulevard Tonnelle, 37000 Tours, France ² Université François Rabelais de Tours, CHRU de Tours, France ³ UMR Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France

⁴ Correspondence address. Tel: +33-247-474-746; Fax: +33-247-478-484; E-mail: royere@med.univ-tours.fr

 BACKGROUND: Whether extended culture allowing selection of embryos with high development potential has any advantage over cleavage-stage embryo transfer remains a matter of debate. Among the currently unsolved questions, the cumulative delivery rate resulting from fresh and frozen embryo transfers needs to be taken into account in both strategies. The aim of our study was, therefore, to compare the efficacy of single embryo transfer either on Day 2 or on Day 5/6 combining fresh and frozen embryo transfers.

METHODS: A prospective study including 478 couples assigned on a voluntary basis to undergo elective single embryo transfer (eSET, n = 243) on Day 2 or single blastocyst transfer (SBT, n = 235) on Day 5/6 was performed. The primary outcome measurement was the cumulative delivery rate including fresh and frozen–thawed cycles in both groups.

RESULTS: The delivery rate per cycle following fresh embryo transfer was significantly higher in the SBT group compared with the eSET group (P < 0.01). Conversely, frozen embryo and/or blastocyst transfers tended to result in a higher number of deliveries in the eSET compared with the SBT group. Altogether, the cumulative delivery rate per couple, including fresh and frozen embryo transfers, was similar between the two groups (37.9% versus 34.2% in the SBT and eSET groups, respectively).

CONCLUSIONS: The observed cumulative delivery rates in this study do not allow us to take a position in favor of SBT or eSET. An improvement in blastocyst cryopreservation may change this attitude.

Key words: blastocyst/cleavage-stage embryo/cryopreservation/cumulative delivery rate/single embryo transfer

Objective