The UK's National Institute for Clinical Excellence (NICE) has published the second draft of its proposed guidelines for infertility treatment provided by the National Health Service (NHS). It recommends that infertile couples meeting certain criteria should be offered up to three IVF attempts using fresh embryos, with the possibility of further treatment cycles using frozen embryos. The proposals could signal an end to the 'postcode lottery' provision of IVF, which at present varies greatly throughout the UK.

The guidance states that couples should be offered state-funded infertility treatment if the woman is between 23-39 years old, and if there is either a diagnosed cause of infertility, or at least three years of unexplained infertility. It also recommends that women under 23 may be offered treatment if the couple have an unequivocal need for IVF treatment, such as prior treatment for cancer, very poor semen quality or Fallopian tubal blockage. NICE also recommends that no more than two embryos per cycle of IVF treatment are transferred to the woman's womb, to reduce the risk of multiple pregnancy.

The infertility patient support group CHILD has welcomed the production of the guidelines: 'The emotional impact of infertility on a couple's life is devastating, and finding themselves unable to access the treatment they need on the NHS causes further distress at an already very painful time in their lives' said spokeswoman Sheena Young. Fertility clinic director Simon Fishel also welcomed the proposals, saying that the UK has been 'the poor relation' in IVF provision compared to other western countries. But he also cautioned that it could take several years before the recommendations are implemented. The guidelines are now available on NICE's website (see link below), and people are invited to send their views until 22 September 2003, Anne-Toni Rodgers of NICE told the BBC. The final draft is due to be published in February 2004.
[ Full Article ]

Preimplantation genetic screening (PGS) for aneuploidy was first reported by Verlinsky et al (1995) and Munne et al (1995). Both of these initial studies analysed polar bodies. The aim of the technique is to help determine the best IVF embryo for transfer on the grounds of the polar body or embryo's chromosomes, by performing biopsy and analysis of the chromosomes using fluorescent in situ hybridisation (FISH). There have been hundreds of papers on the use of PGS. It is well known that for patients with advanced maternal age, there is an increased risk of chromosome abnormalities in the embryos they produce. Therefore this has been the main indication for PGS, but other indications include repeated IVF failure, repeated miscarriage (with normal karyotypes in the parents) and severe male factor infertility. As with many new technologies brought into the IVF clinic, there has been little evidence-based medicine to show that PGS increases delivery rates. At the late breaking news at the 2007 European Society of Human Reproduction and Embryology (ESHRE) annual meeting, Mastenbroek reported that their randomised controlled trial (RCT) on PGS showed that the treatment group had a lower delivery rate compared to the control group (Mastenbroek et al, 2007). Since then the debate about the validity of PGS has been rife. 

In 2007, as chair of the ESHRE PGD Consortium, I was asked if the Consortium steering committee could write a position statement on the use of PGS. The steering committee could not come to a consensus and so a position statement was never issued. The majority of the committee felt that further RCTs were required to convince the world whether PGS did or did not improve delivery rates (Harper et al, 2008). Since 2007, the British Fertility Society (BFS), the American Society for Reproductive Medicine (ASRM) and the American College of Obstetricians and Gynecologists (ACOG) have all issued statements that PGS should not be offered clinically. Several times at ESHRE 2009, I asked the audience how many were routinely performing PGS and many groups said they were.

There are now nine RCTs applied to both good (Staessens et al, 2008, Meyer et al, 2009, Jansen et al, 2008, Mersereau et al, 2008) and poor (Staessens et al, 2004, Stevens et al, 2004, Debrock et al, 2007, Hardarson et al, 2008, Mastenbroek et al, 2007) prognosis patients which have all shown that PGS has not improved the delivery rate, compared to a control group, and some of these studies show it has decreased the delivery rate. Almost all of these studies have been applied to cleavage stage embryos and FISH to study 5-12 chromosomes except Jansen et al (2008) who performed trophectoderm biopsy. Performing the biopsy at cleavage stages has a biological problem as this is the stage when human embryos show high levels of chromosome abnormalities (Harper et al, 1995, Munne et al, 1995) and so analysis of one cell from these embryos is not representative of the rest of the embryo. Many embryos are 'mosaic' - different cells may have different chromosomal complements - which may well self correct to give a normal embryo, but mosaicism creates a problem for PGS.

I reported the latest data of the ESHRE PGD Consortium at ESHRE 2009 in Amsterdam. The data shows that the majority of PGD cycles reported to the Consortium are for PGS (more than all of the other indications added together), but the latest data collection only includes cycles performed to the end of 2007, and so we have yet to see the 'Mastenbroek' effect. It will be interesting to see what the next data collection shows.

At the 2009 ESHRE meeting, I organised a post-congress course on the use of arrays in PGS. Several groups and companies reported on their development and clinical application of array CGH (comparative genomic hybridisation - which just looks at the chromosomes) or SNP arrays (single nucleotide polymorphism - which can look at the chromosomes and genes) for PGS. I summarised the session with three questions: 1) Are the arrays validated? 2) At what stage should we do the biopsy? 3) Have we done the necessary RCTs to determine if PGS will result in improved delivery rates? 

The arrays are being validated using a variety of single cells from normal and aneuploid cell lines and also polar bodies, blastomeres and trophectoderm cells. Cleavage stage biopsy is a good option for PGD for inherited disorders as it allows analysis of the paternal and maternal genes/chromosomes and a fresh transfer, but it may not be the optimal stage to biopsy for PGS (as described above). Therefore the third question needs to be answered by performing RCTs on either polar bodies (first and second) or trophectoderm. 

Also at the 2009 ESHRE conference, Professor Joep Geraedts, the

outgoing chairman, announced that ESHRE would be undertaking its first ever

clinical trial - a two stage study developed to assess the efficacy of PGS using polar body biopsy and analysis of 24 chromosomes using array technology. The first part of the study aims to determine if the technique of polar body biopsy and analysis of 24 chromosomes by array CGH is feasible. Two centres that have extensive polar body and PGS experience (Markus Montag, Hans van der Ven, University of Bonn, Germany and Cristina Magli and Luca Gianaroli, SISMER, Italy) are analysing the first and second polar bodies from a variety of patients who have agreed to be in the study. Any abnormal oocytes will also be examined to see whether the polar body abnormalities can really be used to predict that the oocyte will be abnormal. If the proof of principle part of the study is successful, the second part of the study will involve a multi centre RCT involving at least six centres in different EU countries, to determine if PGS gives an increased delivery rate in women of advanced maternal age. It was decided to run the trial on polar bodies as this will allow for a fresh transfer, the biopsy is less invasive and hopefully the results will be more reliable than cleavage or blastocyst biopsy as mosaicism will not affect the results. Dagan Wells (Oxford) is developing a blastocyst biopsy clinical trial for PGS with vitrification of the biopsied blastocysts to give time for the analysis. 

The UK's Human Fertilisation and Embryology Authority (HFEA) has reviewed the regulation of PGS and now no longer require clinics to offer PGS just for the criteria stated above (advanced maternal age, etc). Instead, they have placed the onus on clinics, requiring them to validate the use of PGS for each category of patient and to warn patients that PGS is an unproven technique in need of further research. If the RCTs show no improvement in delivery rates, it may be time to put PGS behind us, but if they show that PGS improves delivery rates this will be a major step forward for IVF treatment. We need to learn from the PGS experience to ensure that we rigorously test new techniques before we introduce them into clinical practice. 

References

1. Debrock S, Melotte C, Vermeesch J, et al. Preimplantation genetic screening (PGS) for aneuploidy in embryos after in vitro fertilization (IVF) does not improve reproductive outcome in women over 35: a prospective controlled randomized study. Fertil Steril 2007; 88:S237.

2. Hardarson T, Hanson C, Lundin K, et al. Preimplantation genetic screening in women of advance maternal age decrease in clinical pregnancy rate: a randomized controlled trial. Human Reprod 2008; 23:2806-2812.

3. Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 1995; 15:41-49.

4. Harper J. Sermon K, Geraedts J, et al. What next for preimplantation genetic screening? Hum Reprod 2008; 23:478-480.

5. Jansen RP, Bowman MC, de Boer KA, et al. What next for preimplantation screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy. Hum Reprod 2008; 23:1476-1478.

6. Mastenbroek S, Twisk M, van Echten-Arends J, et al. In vitro fertilization with preimplantation genetic screening.

N Engl J Med 2007; 357:9-17.

7. Mersereau JE, Pergament E, Zhang X, et al. Preimplantation genetic screening to improve in vitro fertilization pregnancy rates: a prospective randomized controlled trial. Fertil Steril. 2008;90:1287-9

8. Meyer, L, Klipstein, S, Hazlett, W et al. A prospective randomized controlled trial of preimplantation genetic screening in the "good prognosis" patient. Fertil Steril 2009; 91:1731-1738.

9. MunnÈ S, Sultan KM, Weier HU, et al. Assessment of numeric abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human embryos before transfer. Am J Obstet Gynecol. 1995;172(4 Pt 1):1191-9.

10. Staessen C, Platteau P, Van Assche E, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advance maternal age: a prospective randomize controlled trial. Hum Reprod 2004; 19:2849-2858.

11. Staessen C, Verpoest W, Donoso P, et al. Preimplantation genetic screening does not improve delivery rate in women under the age of 36 following single-embryo transfer. Hum Reprod 2008; 23:2818-2825.

12. Stevens J, Wale P, Surrey ES, Schoolcraft WB. Is aneuploidy screening for patients aged 35 or over beneficial? A prospective randomized trial. Fertil Steril 2004; 82(Suppl. 2):249

12. Verlinsky, Y, Cieslak, J, Freidine,M et al. Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Mol Hum Reprod 1995;1:265-269.

[ Full Article ]

A report published by the US Centers for Disease Control and Prevention (CDC) shows that the younger a woman is when she uses assisted reproductive technology (ART), the more likely she is to become pregnant and have a live birth using her own eggs. The report defines ART as procedures in which both egg and sperm are handled in a laboratory, and says the majority of ART treatments its data include refers to IVF.



The CDC's annual report, released last week, used data for the year 2002 collected from 391 of the 428 fertility clinics in the US. The report, called '2002 Assisted Reproductive Technology Success Rates', showed that, in 2002, 45,751 live births were achieved from 115,392 ART procedures performed in the US. This was an increase from the previous year's figures, when there were 40,687 live births from 107,758 treatments. Overall, the per-cycle ART success rate in 2002 was 35 per cent, compared to 28 per cent in 1996.



The 2002 data show that 37 per cent of women who undergo ART using their own, fresh eggs when they are below the age of 35, had a live birth. This is compared with 31 per cent of women aged between 35 and 37; 21 per cent of women aged between 38 and 40; 11 per cent of women aged between 41 and 42 and just four per cent for women older than 42. However, the report also showed that the age of the woman undergoing ART had 'little effect' on success rates if donated eggs were used. In 2002, the live birth rate for all ART procedures where donated eggs were used was 50 per cent, with the success rate varying only slightly between age groups.



Victoria Wright, one of the authors of the CDC report, said that the data show that 'women in their 20s and early 30s who used ART had the most success with pregnancies, and single live births'. But, she added, 'success rates declined steadily once a woman reached her mid-30s'. She said the figures should act as 'a reminder that age remains a primary factor with respect to pregnancy success, and younger women have greater success than older women, even with technology'.
[ Full Article ]

The US Food and Drug Administration (FDA) is introducing new rules about who can donate sperm. Men that have had homosexual sex within the five years prior to them wanting to make an anonymous sperm donation will be prevented from doing so, as the FDA says that gay men are collectively more likely to be HIV-positive than other men. However, men who have had male sexual partners within the past five years would not be prevented from donating sperm to a friend or family member. Critics accuse the FDA of stigmatising gay men rather than putting in place a screening process that focuses on high-risk sexual behaviour by any potential donors, gay or straight.



The new rule is part of a set of regulations on tissue donation, which require tissue banks to test donors and donated tissues for HIV, hepatitis B, hepatitis C, syphilis and other diseases, as well as sexually transmitted infections like chlamydia and gonorrhoea, particularly for sperm and egg donation. Tissue banks will also be required to ask donors about their risk factors for such diseases. The only exceptions exist when cells or tissues are being transplanted back into the person who donated them or to their sexual partner or for people who are repeat anonymous sperm donors.



Gay rights groups have condemned the new regulations, saying that they are discriminatory and would be would be difficult to enforce. Ronald Johnson, associate executive director of Gay Men's Health Crisis, said the regulations lacked scientific merit: 'it is bad science because it injects a false sense of security and the impression the government is doing something, when in fact they are not taking the more effective way to do proper screening and protection', he said. He added that 'we feel that there are adequate and reliable screening procedures that ought to be in place rather than a blanket rule that discriminates against sexually active gay men as a whole category'.



Leland Traiman, director of Rainbow Flag Health Services, a sperm bank based in California, said: 'Under these rules, a heterosexual man who had unprotected sex with HIV-positive prostitutes would be OK as a donor one year later, but a gay man in a monogamous, safe-sex relationship is not OK unless he's been celibate for five years'. Instead, he proposed that donors could be tested for HIV at the time of donation, then the sperm frozen for six months and the donor tested again six months later to ensure sperm safety. But a spokeswoman for the American Society for Reproductive Medicine said that 'you can't be too careful' with anonymous sperm donation, adding 'our concern is for the health of the recipient, not to let more and more people be sperm donors'. A spokesperson for the FDA said that the new regulations are based on 'scientific consideration'.

[ Full Article ]

A Belfast woman has given birth to the first 'saviour sibling' conceived in the UK: a baby girl who could help treat her seriously ill three-year-old brother. In September 2004, the Human Fertilisation and Embryology Authority (HFEA) granted Joe and Julie Fletcher permission to have a tissue-matched baby to help treat Joshua, who has an incurable blood disorder. Following successful treatment at the Assisted Reproduction and Gynaecology Centre in London, Mrs Fletcher gave birth to daughter Jodie on 14 July.



Joshua Fletcher has Diamond Blackfan anaemia (DBA), a rare blood condition that could be cured with a blood stem cell transplant from a tissue-matched donor. Having failed to find a suitable existing donor, his parents applied to use preimplantation genetic diagnosis (PGD) to conceive an IVF baby who would be able to provide Joshua with compatible umbilical cord blood cells. If transplanted to Joshua, these cells could enable his body to produce its own healthy red blood cells. Doctors collected cord blood cells immediately after the birth of Jodie Fletcher, who should be a perfect genetic match for her brother.



The transplant will not happen for at least another six months, however, since doctors must first wait and see if Jodie is also affected by DBA. Some cases of DBA are caused by a mutation in a gene called RPS19, but for most the trigger remains unknown. For this reason it was not possible for the Fletchers to use PGD to select an embryo that would definitely be free from the condition. However, the couple are optimistic that the stem cell treatment will take place next year. 'My instinct tells me that the transplant will go ahead', said Mr Fletcher, adding 'the thought of having a Joshua who doesn't have DBA fills me with joy. It's like someone telling you that in 12 months you are going to win the lottery'. But for the time being, the family are simply enjoying their new baby. Mrs Fletcher said that Jodie was 'just as important for herself', adding 'she is another baby in our family, one we wanted anyway'.



The HFEA's decision to allow the Fletchers to conceive a tissue-matched baby followed the authority's recent policy change in this area, allowing couples to use PGD for testing IVF embryos solely to check their suitability as a potential cord blood donor for an existing sick child. Although Jodie Fletcher is the first potential saviour sibling conceived in the UK, another British baby has already helped his older brother in this way. In 2002, the HFEA turned down a request from the Whitaker family, who were also seeking to use PGD to conceive a tissue-matched baby to help a sibling with DBA. Michelle and Jayson Whitaker later travelled to Chicago to conceive their son James, born in June 2003, whose umbilical cord blood has now been successfully used to help treat their son Charlie.

[ Full Article ]

The University of Kentucky now offers three graduate degree options in the Reproductive Sciences and Reproductive Laboratory Science, including a: 1)  multidisciplinary Ph.D. in Reproductive Sciences; 2) master’s degree in Reproductive Laboratory Science; and 3) a Graduate Certificate in Reproductive Laboratory Science.  Please refer to:  http://www.mc.uky.edu/healthsciences/academic/index.html for complete information, including program requirements, curricula, course schedules, and procedures for application. 

 

The Ph.D. is multidisciplinary in that coursework is  completed in both the College of Medicine’s Interdisciplinary Biomedical Sciences curriculum and the College of Health Science’s Reproductive Sciences program, as well as other UK departments (e.g. statistics).  The program is unique in that students have the option of completing the clinical curriculum in embryology/andrology as they progress through the basic science curriculum and complete dissertation research.  Students may complete research in the laboratory of a faculty member, who is an active member of the University of Kentucky Reproductive Sciences Center, which is currently moving through the University approval process.  It is anticipated that the university-wide Center will receive final  approval in spring/summer, 2010. A limited number of competitive fellowships are available for qualified Ph.D. students. 

 

The Master of Science is now a one-calendar-year curriculum (August-July).  Although the Graduate Certificate is available to qualified individuals holding a B.S. in science or medical laboratory science, the certificate is intended for individuals already holding an advanced degree.  Scholarships for the M.S. are limited and highly competitive. 

 

Students enrolled in the clinical curriculum (M.S., Graduate Certificate, and Ph.D. with clinical option) complete practica at assisted reproductive technology centers throughout the United States, with the possibility of international internships.   All clinical faculty directing students, are appointed as clinical faculty by the University of Kentucky.  ART practitioners throughout the U.S. also contribute to  didactic and student laboratory instruction to ensure that the curriculum is current and in line with clinical practice.

 

Applications to both the Graduate School and the specific graduate program is required for the Ph.D., the M.S. and the Graduate Certificate.  Refer to:  http://www.gradschool.uky.edu/.  This site will also direct you to funding that is available through the Graduate School. 

 

The deadline for applications for U.S. citizens/residents is April 15, 2010. 

 

 

Please feel free to contact me if you are interested in one of the graduate programs after reviewing the information on the websites.

 

 

Doris J. Baker, Ph.D., HCLD(ABB), MT(ASCP)

Professor, Center of Excellence in Reproductive Sciences

Director of Graduate Studies, Reproductive Sciences University of Kentucky

126E CTW Building

900 South Limestone

Lexington, KY 40536-0200

Telephone:  (859) 218-0854

Fax: (859) 323-8957

E-mail: [email protected]

[ Full Article ]

Authors: World Health Organization, Department of Reproductive Health and Research
Number of pages: 287
Publication date: 2010
Languages: English
ISBN: 978 92 4 154778 9 

---

Full text

---

In press. This publication will be for sale.

OVERVIEW

Semen analysis may be useful in both clinical and research settings, for investigating male fertility status as well as monitoring spermatogenesis during and following male fertility regulation and other interventions. This manual provides updated, standardized, evidence-based procedures and recommendations for laboratory managers, scientists and technicians to follow in examining human semen in a clinical or research setting. Detailed protocols for routine, optional and research tests are elaborated. The fifth edition includes new information on sperm preparation for clinical use or specialized assays and on cyropreservation, an expanded section on quality control in the semen analysis laboratory and evidence-based reference ranges and reference limits for various semen characteristics. The methods described are intended to improve the quality of semen analysis and the comparability of results from different laboratories.

 

[ Full Article ]

Koji Matsuura a,b, Nobuyoshi Hayashi c, Yuka Kuroda a,b, Chisato Takiue c, Rei Hirata c, Mami Takenami a, Yoko Aoi c, Nanako Yoshioka c, Toshihiro Habara c, Tetsunori Mukaida d, Keiji Naruse a,*

a Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan; b Research Core for Int erdisciplinary Sciences, Okayama University, Okayama, Japan; c Okayama Couples Clinic, Okayama, Japan; d Hiroshima HART Clinic, Hiroshima, Japan

* Corresponding author.

E-mail address: [email protected] (K Naruse).

Dr Keiji Naruse graduated from Nagoya University School of Medicine in 1988 (MD) and received his PhD in medicine from Nagoya University in 1992. He was an associate professor at Nagoya University from 1999 to 2005 and is currently a chairman and professor of Cardiovascular Physiology at Okayama University Graduate School of Medicine. He was a visiting professor at Harvard Medical School from 1998 to 2001. He has been working in the fields of mechanobiology of circulation, reproduction, and sensory systems.

Abstract

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression and friction force in the Fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, the tilting embryo culture system (TECS) was developed. The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blas- tocyst stage and significantly increased the cell number of mouse blastocysts (P < 0.05). Although not statistically significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared with static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low-quality embryos and those embryos cultured under suboptimal conditions (P < 0.05). The TECS is proposed as a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to those in the Fallopian tube.

RBMOnline © 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Declaration: Keiji Narusee launched the bio-venture company, Strex Inc., in 2003 and serves as a Chief Scientific Officer. The other authors report no financial or commercial conflicts of interest.

KEYWORDS: blastocyst, embryo development, mechanical stimuli, shear stress, tilting embryo culture system

Introduction

Mammalian embryos are transported to the uterine cavity through the Fallopian tube during cell cleavage, blastomere and blastocyst development (Eddy and Pauerstein, 1980; Halbert et al., 1976). In conjunction with ciliated epithelium, the Fallopian tube acts as a peristaltic pump due to phasic contraction of the smooth muscle in the wall to transport the embryo (Lyons et al., 2006; Zervomanolakis et al., 2007). Based on the movement of cilia and the similar size between the tubal lumen of ampulla and isthmus (Table 1) and the diameter of the embryo (0.1 mm), fertilized oocytes may be subject to a mechanical influence from the Fallopian tube lumen, such as compression and shear stress from the tubal fluid. It is postulated that these mechanical actions in the Fallopian tube might play an important role in embryo development. However, conventional in-vitro static culture conditions do not mimic mechanical stimuli to embryos. Providing mechanical stimuli to developing embryos in-vitro, similar to that experienced in the Fallopian tube, may improve development.

From the 1980s, some groups reported in-vitro non-static culture results (Nagai et al., 1993; Staigmiller and Moor, 1984). However, these reports did not discuss the precise effect on embryo development from cleaved embryo to blastocyst. Recent papers have reported that a microfluidic dynamic embryo culture system with media flow improved mouse embryo development (Cabrera et al., 2006). The report proposed the importance of the physical/mechanical environment on embryo development. Because the systems are complex to handle, clinical application of the culture system is quite difficult. In addition to ease-of-use, another consideration regarding mechanical stimulation during embryo development is avoiding excess stress. Excess mechanical stimuli would damage embryos. It was found that shear stress over 1.2 dyn/cm² caused lethality within 12 h for blastocysts (Xie et al., 2006). Over-handling of embryos, such as excess pipetting, caused elevation of phosphorylated stress- activated protein kinase and may cause rapid transient changes in hundreds of proteins and mRNA (Xie et al., 2007).

In order to construct a dynamic culture system that can apply a mechanical stimulus and can be easily adapted to conventional static culture platforms, the tilting embryo culture system (TECS) was developed. Placing a conventional culture dish or plate on a tilted plate makes embryos in culture move along the bottom of the dish by gravity. Animal model experiments using TECS were first conducted, for the in-vitro culture of mouse 2-cell-stage embryos. A subsequent study using thawed human embryos cultured on TECS was also performed.

Materials and methods

Observation of embryo motion and shear stress calculation

To estimate the shear stress applied to embryos, mouse embryo motion in the microdrop on the tilting plate of a prototype TECS was observed by objective lens (20x) attached to the bottom of the tilting TECS plate. To estimate the flow velocity of media in the microdrop, this study observed the motion of the microspheres (0.5–5 μm diameter) in the microdrop covered with mineral oil by tilting inverted microscope (Eclipse, Nicon, Tokyo, Japan), particularly focusing on the motion of microspheres at the centre of the bottom where embryos in the microdrop were moving. When the microscope was tilted, the particles moved in the same direction as the leaning microscope. The average particle velocities, which were considered as the velocity of the medium in the microdrop, were calculated. Under the holding the tilt and static conditions, Brownian motion of the particles was dominant. Images of moving embryos and microspheres were recorded by a charge-coupled device camera connected to a personal computer. The frame rate of the recording was 30 frames/s.

The observed maximum velocity of the embryos and the velocity of the medium were V_E and V_M, respectively. The velocity to estimate maximum shear stress (V_SS) was calculated from the difference between V_E and V_M.

V_SS =V_E -V_M

 The maximum shear stress applied to embryos during the tilting was calculated from the following equation:

Shear stress = 6µrV_SS =4µr² = 6V_SS dyn/cm²

where µ is the viscosity of the medium and r is the radius of the embryo.

In an attempt to provide as accurate an estimate as possible regarding sheer stress experienced by embryos, this study attempted to determine the flow velocity of fluids using microspheres. The above equations are applicable in the flow condition. Generally, the effect of Brownian motion during flow can be neglected for the shear stress calculation. However, it would be difficult to determine accurate fluid velocity by particle image velocimetry. Although forces such as friction come into play with this approach, it provides a rough estimation for the final calculations.

Embryo motion in extirpated mouse oviducts was observed as follows. Female ICR mice (8–12weeks old; Charles River Japan, Yokohama, Japan and Shimizu Laboratory Supplies, Kyoto, Japan) were injected with 5 IU pregnant mare’s serum gonadotrophin (Aska Pharmaceutical, Tokyo, Japan), followed by 5 IU human chorionic gonadotrophin (Aska Pharmaceutical) 48h later and mated with males. Successful mating was determined the following morning by the detection of a vaginal plug. Two days after the mating, the oviduct was extirpated with embryos and the oviduct was sandwiched between a cover slip and a glass slide to observe the embryo in the oviduct. The velocity of the embryo in extirpated mouse oviducts was calculated from the movie of the embryo motion. Animal use protocols were approved by Okayama University animal investigation committee.

Specification and motion programmes of TECS

TECS (Strex, Osaka, Japan) is an electrical device with a power cord that is designed to be used in a humidified incubator. This device consists of a control unit (Figure 1A, upper) and a motor unit with a tilting plate (Figure 1A, lower). Four-well chambers and/or dishes are set on the plate. The TECS motor unit is controlled and DC-powered by a cord connected to the control unit through an access hole in the incubator. If the incubator does not have an access hole, a flat-type cable extends tightly through the door of the incubator in order not to alter temperature, gas concentration and humidity inside the incubator. The motor unit is waterproof. The controller outside the incubator puts in the motion parameters.

A representative motion programme of TECS is shown in Figure 1B and C. The TECS can control the parameters of the uniform radial velocity (Vr), the maximum tilt angle (θr) and the holding time at the maximum tilt angle (Th). The tilting time (Tr) in seconds is calculated from 2θr/Vr. First, the plate is tilted to the positive maximum tilt angle (+θr) (M1). Second, the tilting plate is held for Th with no motion (M2). Third, the plate is tilted to the negative maximum tilt angle (−θr) (M3). Last, the tilting plate is held for Th with no motion (M4). The TECS motion cycle is summarized in Figure 1C. The cycle continues until the power is turned off.

To apply conventional culture dishes/approaches to the TECS, spill out of the mineral oil should be prevented. The angles at which mineral oil begins to spill out are different according to the size of the dishes. The mouse embryo experiments used dishes of 35 mm diameter and maximum tilt of 20 degrees. However, in the human embryo study, the plate could not be tilted over 10 degrees due to the use of dishes of 60 mm diameter.

The maximum tilt angle that caused mineral oil to spill out was surveyed and the limit of the maximum tilt angle was approximately 20 degrees in the microdrop setup. Furthermore, excess uniform radial velocity also induces spill out of the mineral oil. The minimum radial velocity at which the mineral oil spilt from the 35 mm dish was 240 degree/s when the maximum tilt angle was 20 degrees. The oil did not spill out when the tilt angle was 10 degrees. It is necessary to increase the maximum tilt angle and radial velocity in order to move embryos in the microdrop. However, this study set the maximum tilt angle and radial velocity so that they did not result in the spilling out of the mineral oil but still allowed the observation of embryo motion in the video rate recording. Thus, the maximum tilt angle was 10–20 degrees and the radial velocity was approximately 1 degree/s.

Tilting mouse embryo culture

Frozen 2-cell-stage embryos of ICR mouse (Arc Resources, Kumamoto, Japan) were thawed and cultured in 50 and 500 μl, respectively, of potassium simplex optimized embryo culture medium (Daiya Shiyaku, Tokyo, Japan). The culture medium was covered with mineral oil and incubated for 3 days in a humidified environment of 5% CO₂ in air at 37°C. In the TECS group, the mouse embryos were cultured at a maximum tilt angle of 20 degrees with a holding time of 1 min. The plate was rotated at 1 degree/s to reach a total tilt of 20 degrees. To count cells in the mouse blastocyst, the cells were stained with Hochest 33342, as previously reported (Hardy et al., 1989; Mottla et al., 1995). The stained blastocysts were observed by confocal microscopy (FV-1000; Olympus, Tokyo, Japan) and a 3D image was constructed.

Human embryo study

The human embryo study used 3- to 11-cell-stage embryos frozen by the slow method 3 days after collection of the oocytes from May 2000 to December 2004 (Cohen et al., 1985) and extended the prospective study of 220 fertilized human embryos that were to be discarded with consent after pregnancy of patients. This study was approved by the ethics committee of the Okayama University Graduate School of Medicine.

The frozen embryos were thawed with THAW-KIT 1 (Vitrolife, Gothenburg, Sweden) and the viability of the thawed embryos was approximately 80%. Once thawed, human embryos were cultured in a 20 ll microdrop of Global medium (LifeGlobal, Ontario, Canada) covered with mineral oil for 48 h. The thawed embryos with morphologically regular cleavage were divided so that there would be the same percentage of embryos with regular cleavage (33%) in both the TECS group and the control group. The viable cell number of the thawed human embryos was from three to 11. The dishes were placed on the tilting plate of TECS in a 50 l multi-gas incubator (ASTEC, Fukuoka, Japan). The thawed human embryos were cultured at a 10 degree tilt for 10 min. The radial velocity of the tilt was 1 degree/s (V_r).

The blastocysts were morphologically evaluated according to the classification of Gardner et al. (2000). The cells in the human blastocyst were stained with Hochest 33342, the blastocysts were observed by confocal microscopy (FV-1000 Olympus, Tokyo, Japan) and a 3D image was constructed.

Statistical analysis

Chi-squared test and Student’s t-test were used to determine differences in the blastocyst development rate and in the number of cells in the blastocysts between the groups, respectively. A P-value < 0.05 was considered significant.

Results

Observation of embryos in motion by TECS and estimation of shear stress

To obtain detailed information on the embryo motion, the blastomeres were observed on the TECS during tilting and holding. Figure 2 shows cropped images of the mouse embryos in motion. Between each 10 degree tilt (M1 and M3 in Figure 1B), the TECS plate was tilted at 1 degree/s. The motion of the medium was observed in M1 and M3 (Figure 1B). The embryo moved at a velocity of 1.5 mm/min on average (Figure 2A and B), which gave rise to shear stress of 7.0 x 10^-3 dyn/cm² at the bottom of the centre in the microdrop at a velocity of 0.1 mm/min. When the TECS plate was held at a 10 degree tilt (M2 and M4 in Figure 1B), the embryo slid at a velocity of 0.03 mm/min (Figure 2C, D), which gave rise to a shear stress of 1.5 · 10^–4 dyn/cm² in the medium, where the motion of medium at the centre of the bottom was neglected in the motion phases. These values are summarized in Table 2.

Mouse embryo development

Table 3 shows the blastocyst development rates from 2- cell-stage mouse embryos cultured under several conditions. As shown in Figure 3, the blastocyst development rate was 79% for 10 embryos in a 50 ll microdrop using the TECS, which was not significantly different to the static culture controls (75%). Because mouse blastocyst development rate is reported to be influenced by the number of embryos in the culture medium (Melin et al., 2009) and in order to enhance the statistical difference between TECS and control, the number of embryos in the microdrop were deduced and the medium volume-to-embryo ratio, which might simulate poor development condition and lead to low-quality human embryos, was increased. The blastocyst development rate for four–six embryos in a 50 ll microdrop was less than that of 10 embryos, although in this poor condition, TECS significantly improved the blastocyst development rate (TECS 59% (n = 145) versus control 46% (n = 151); P < 0.05). When 10 embryos were cultured in 500 ll medium, the blastocyst development rate decreased to 27% (n = 101) in the static control, whereas for those cultured in the TECS it was significantly higher (42% (n = 99); P < 0.05)). The number of cells in the blastocysts cultured using the TECS was (mean ± SEM) 77 ± 4 cells (n = 34), while that of the control was 66 ± 4 cells (n = 26), as shown in Figure 4. There was a significant difference in the average cell number between the two groups (P < 0.05).

Human embryo development

As TECS enhanced blastocyst development rate in mouse embryos, experiments were then conducted on thawed human embryos. The development rates to the blastocyst stage of the TECS and control groups were 53% and 45%, respectively (Table 4).

Due to the insufficient sample number of thawed human embryos, the study investigated the cell number of the developed blastocysts instead of the blastocyst development rate. Figure 5 shows the comparison of average cell numbers in human blastocysts developed by the TECS and control groups. The mean cell number of developed blastocysts at day 5 by the TECS was 43±3 cells (n=24), while that of the control was 34 ± 3 cells (n = 18). There was a significant difference in the average cell number between the two groups (P < 0.05).

Discussion

The Fallopian tube is a multifunctional organ, involved in receiving ovulated oocytes, providing a suitable environment for fertilization and early development and transporting embryos to the uterus. Here, light is shed on the mechanical properties of the Fallopian tube that might influence early development of embryos during culture. Early studies showed that the Fallopian tube is a mechanically active organ and may have influence on development due to: (i) shear stress by a tubal fluid flow; (ii) compression by peristaltic tubal wall movement; (iii) buoyancy; and (iv) kinetic friction force between embryo and cilia. Punctuated velocities of maxima from 0.39 to 1.8 mm/min have been observed in rat oviducts for microspheres emulating the size of embryos (Xie et al., 2006). Inappropriate culture conditions could be detrimental. Indeed, embryos sense shear stress and development is compromised (Xie et al., 2006, Xie et al., 2007). Because shear stress is a function of the velocity of the embryo and the flow and because the contribution is important, it is propose that the velocity of embryos should be made similar to those in the oviduct. Similarity in the velocities of the mouse embryos was found but those of other species were not observed. The velocities in-vivo and ex-vivo should be investigated for optimization.

The observed velocity of mouse embryos on a TECS culture plate was of a similar order to those proposed in the oviduct. The embryo motion in the microdrops on the TECS plate relates directly to the plate motion. The observed embryo images on the TECS plate suggest that the velocities and shear stresses of embryos (Table 2) in the TECS plate and the oviduct are similar. This comparative experiment suggests that the TECS can apply physiological mechanical stimuli to mouse embryos. In the case of human embryos, the embryo velocity in the microdrop on the TECS plate was the same order (approximately 1 mm/min) as those of mouse embryos and below 1.2 dyn/cm². Therefore, the TECS could apply physiological stimuli without an excess amount of shear stress that might cause damage to the embryos. Furthermore, the detrimental or beneficial shear stress would be different at each developmental stage and future experiments will address this.

Embryo motion in fluid is affected not only by species differences but also the environment of the embryos. For example, when cumulus cells are coated with zona, embryos with the cells stick on the bottom of the dish. To optimize the parameters (θr and Vr) for each embryo condition, the relationship between the motions in fluid and physical characteristics of embryos (such as density, dimension and zona surface structure) should be considered.

The TECS significantly improved development in low-quality human embryos and suboptimal culture conditions in mouse embryos. The significance of the improvement by the TECS was dependent on the number of mouse embryos in the medium. As mentioned in the results, the blastocyst development rate significantly improved in the case of four–six mouse embryos in the microdrop. In the case of 10 embryos in the microdrop, blastocyst development rates of the TECS and control groups were 75% and 79%, respectively, and the difference was not significant. A higher number of embryos in the medium can improve the blastocyst development rate. According to previous reports that the concentration and production of autocrine and/or paracrine factors enhance mouse embryo development (Contramaestre et al., 2008, Kawamura et al., 2005), a higher number of embryos in the medium can improve the blastocyst development rate. The results in Figure 3 demonstrate that blastocyst development rate was significantly improved by the TECS when using a lower ratio of mouse embryo to media volume. This may be due to diffusion of growth factors and/or waste products, which would be facilitated by the TECS motion in the mouse embryo culture. However, mechanical stimuli could also be beneficial. Indeed, in bone and endothelial cells, down-stream transcription factors in the nucleus have been shown to be activated by mechanical stimuli, such as shear stress and mechanotransduction, and gene transcription and DNA syntheses were also activated (Wang and Thampatty, 2006). Due to the enhancement of cell division induced by these activations, cell numbers would be increased without apoptosis.

The results demonstrated that the cell numbers of the mouse and human blastocysts cultured in the TECS were greater than those cultured under control conditions and that TECS can improve the quality of those blastocysts. The increase in cell number of the mouse and human blastocysts suggests that TECS could enhance cell division of human embryos. According to published material (Cui et al., 2008, Xie et al., 2006), negative correlations between the percentage of TUNEL-positive cells and cell numbers in mouse embryos have been suggested. Therefore, although not measured, reduction of necrosis and/or apoptosis may be one explanation for the increased cell number in blastocysts obtained from TECS culture. Future experiments will confirm or refute this theory.

Finally, there are advantages of the TECS in clinical use. Although the culture conditions are different in each assisted reproduction laboratory, a benefit of this system is its ability to be rapidly implemented because it can be adapted to multiple styles of culture dishes/approaches. These results of thawed mouse and human embryo development indicate that the clinical study of embryo culture using the TECS can be extended without problems. To demonstrate the clinical importance of the TECS in human embryo development, a clinical multicentre study is being prepared on human embryo development using the TECS and embryo transfer. The improved quality of developed embryos by the TECS might contribute to enhanced pregnancy rates in clinical practice. As blastocyst cell numbers were increased by the TECS, pregnancy rates resulting from embryos cultured in this system might be improved.

In conclusion, the TECS enhanced blastocyst development rates of mouse embryos after the 2-cell stage and caused a significant increase of cell number in blastocysts. Thawed human embryos after the 3-cell stage tend to show an improved blastocyst development rate when cultured by the TECS. In particular, the improvements made by the TECS were significant in low-quality embryos and suboptimal culture conditions. One possible reason for the improvements could be mechanical stimuli by embryo motion based on the comparison of both mouse and human embryo development results.

 

Acknowledgements

This study was supported by a grant-in-aid for Scientific Research on Priority Areas (No. 17076006 to K.N.) and Special

Coordination Funds for Promoting Sciences and Technology from the Ministry of Education, Science, Sports, and Culture, Japan (to K.M.).

References

Cabrera, L.M., Heo, Y.S., Ding, J., et al., 2006. Improved blastocyst development with microfluidics and Braille pin actuator enabled dynamic culture. Fertil. Steril., S-43.

Cohen, J., Simons, R.F., Edwards, R.G., et al., 1985. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fert. Embryo Transfer 2, 59–64.

Contramaestre, A.P., Sifontes, F., Marin, R., et al., 2008. Secretion of stem cell factor and granulocyte-macrophage colony-stimulating factor by mouse embryos in culture: influence of group culture. Zygote 16, 297–301.

Cui, X.S., Shen, X.H., Kim, N.H., 2008. High motility group box 1 (HMGB1) is implicated in preimplantation embryo development in the mouse. Reprod. Domest. Anim. 75, 1290–1299.

Eddy, C.A., Pauerstein, C.J., 1980. Anatomy and physiology of the fallopian tube. Clin. Obstet. Gynecol. 23, 1177–1193.

Fox, G.J., 2007. The Mouse in Biomedical Research, second ed. Academic Press, pp. 97–98.

Gardner, D.K., Lane, M., Stevens, J., et al., 2000. Blastocyst score affects implantation and pregnancy outcome. Fertil. Steril. 73, 1155–1158.

Halbert, S.A., Tam, P.Y., Blandau, R.J., 1976. Egg Transport in the rabbit oviduct: the roles of cilia and muscle. Science 191, 1052–1053. Hardy, K., Handyside, A.H., Winston, R.M.L., 1989. The human

blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 107, 597–604. Kawamura, K., Fukuda, J., Kumagai, J., et al., 2005. Gonadotropin-releasing hormone I analog acts as an antiapoptotic factor in

mouse blastocysts. Endocrinology 146, 4105–4116. Lyons, R.A., Saridogan, E., Djahanbakhch, O., 2006. The reproductive significance of human fallopian tube cilia. Hum. Reprod. 12, 363–372.

Melin, J., Lee, A., Foygel, K., et al., 2009. In vitro embryo culture in defined sub-microliter volumes. Dev. Dyn. 238, 950–955.

Mottla, G.L., Adelman, M.R., Hall, J.L., et al., 1995. Lineage tracing demonstrates that blastomeres of early cleavage-stage human pre-embryos contribute to both trophectoderm and inner cell mass. Hum. Reprod. 10, 384–391.

Nagai, T., Ding, J., Moor, R.M., 1993. Effect of follicle cells and steroidogenesis on maturation and fertilization in vitro of pig oocytes. J. Exp. Zool. 266, 146–151.

Staigmiller, R.B., Moor, R.M., 1984. Effect of follicle cell on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res. 9, 221–229.

Suarez, S.S., 1987. Sperm transport and motility in the mouse oviduct: Observation in situ. Biol. Reprod. 36, 203–210.

Wang, J.H.C., Thampatty, B.P., 2006. An introductory review of cell mechanobiology. Biomech. Model. Mechanobiol. 5, 1–16.

Xie, Y., Wang, Y., Zhong, W., et al., 2006. Shear stress induces preimplantation embryo death that is delayed by the zona pellucida and associated with stress-activated protein kinase mediated apoptosis. Biol. Reprod. 75, 45–55.

Xie, Y., Wang, Y., Puscheck, E.E., et al., 2007. Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos. Mol. Reprod. Dev. 74, 1287–1294.

Zervomanolakis, I., Ott, H.W., Hadziomerovic, D., et al., 2007. Physiology of upward transport in the human female genital tract. N.Y. Acad. Sci. 1101, 1–20.

Declaration: Keiji Narusee launched the bio-venture company, Strex Inc., in 2003 and serves as a Chief Scientific Officer. The other authors report no financial or commercial conflicts of interest.

Received 16 May 2009; refereed 8 June 2009; accepted 11 November 2009.

 

[ Full Article ]

Researchers at Cornell Medical Center in New York have discovered that commonly prescribed anti-depressants may have the unwanted side effect of drastically lowering male sperm count. Tests were conducted on two men over a two year period, during which time their sperm count changed from normal before taking the anti-depressants, to almost zero after taking the medicines. The sperm count of both men recovered to normal levels once use of the drugs was discontinued.

The men studied were taking anti-depressant medications Citalopram (Cipramil) and sertraline (sold as Lustral in the UK). These drugs are of the same class as market leaders Seroxat and Prozac. Impotence and delayed ejaculation are known to be side effects of these drugs, but this study indicates that there may also be an effect on the nerves in the vas deferens such that sperm are not being transported or released in the ejaculate. Cornell Medical Center found a similar, although less severe effect, on twelve further men and have begun a clinical trial of 30 men taking sertraline.

Lead researcher Dr Peter Schlegel, who presented the research at annual meeting of the American Society for Reproductive Medicine in New Orleans, said, 'These were men with normal sperm counts that went to nearly zero when they were on these antidepressants but returned to normal when they were off them. It's a dramatic effect and it's never been described before. We believe that while it's had a profound effect on these two men, it could be having a significant but more subtle effect on many more men'. Doctors urged men using the drugs not to stop taking them as sudden changes in use may worsen psychiatric conditions, but to consult their GPs if they had concerns.

In research also related to male fertility Australian scientists have reported a link between male infertility and increased risk of testicular cancer. 740 men who attended a Perth infertility clinic over 30 months were given ultrasound scans to check for testicular cancer, of these men five were found to have the condition, which can be hard to detect. This translates to an incidence within this population of a 0.7 per cent lifetime risk of contracting the illness, higher than the usual lifetime risk of 1 in 243. Lead researcher Dr Anne Jequier, addressing the Fertility Society of Australia's Annual Scientific Meeting, called for all infertile men to be given a testicular ultrasound every two to three years in a similar way that mammograms may be routinely offered to older women.

[ Full Article ]

This week, the British media has gone crazy about a newborn baby. His name is James Whitaker and he was conceived in order to provide stem cells for his older brother, Charlie. In the reams of commentary which followed James' arrival into the world, two main ethical issues emerged. The first question is whether it is wrong to create a child in the hope that it will be of some use to another child. The second issue, which follows from the first, is whether it is psychologically harmful to a child to know that it has been born for this purpose.

These questions raise what seem like new dilemmas brought on by the new age of genetics. But, in reality, they are rather old quandaries which many parents have faced before. Couples with a sick child whose health depends upon a matched tissue donation have been known to have another child in the hope that they will be a suitable donor. Whilst this might not be considered the most noble reason for having a child, it is usually regarded as a private family decision. And indeed it was in the case of the Whitakers, whose second child, Emily, was conceived naturally, but who sadly turned out not to be a compatible donor for Charlie. To answer the second question, whether psychological harm will be done to the so-called saviour child, one need only look at Emily, who shows no signs of being neglected or psychologically scarred in any way, despite the fact that her stem cells could not be used to treat her older brother.

If parents have been left to make such judgements privately up until now, why were the Whitakers forced to have their wishes scrutinised by the nation? Because the technique they sought to use is one normally carried in order to avoid the birth of a child with an inherited disease. In the case of the Whitakers, the government regulator, the HFEA, did not consider the reason for having embryo screening to be sufficiently immediate to warrant its use. As chairman, Suzy Leather, told a number of newspapers, 'No one can say for certain what the long-term risks of embryo biopsy are. If there are benefits for the child to be created from the biopsied embryo... the balance of potential harm and potential good falls in a different place than if you're simply biopsying an embryo for the benefit of another person.'

This sounds reasonable enough. But it is important that we do not get safety and morals mixed up in a case like this. If people have difficulty accepting what the Whitakers did, they have to find a way of explaining why they think that. Perhaps people who object to this case really are concerned about the slim chance that the procedure will have damaged James Whitaker in some way. But sometimes safety concerns can be an easy route out of a moral maze. Mere mention of a 'precautionary approach' is often enough to trump all other concerns.

The fact that James did not himself benefit from the embryo screening procedure does make the issue slightly different, but only in the sense that it might have made it a more difficult decision for his parents. Decisions that parents make in relation to their children are often about weighing up risks and benefits and making a judgement which seeks to maximise their health and well-being. The difference with Jayson and Michelle Whitaker is that, because of their unfortunate situation, the stakes were higher.
[ Full Article ]