IVF NEWS.Direct! Journal by iLogy iLogy

January - March 2012

IVF NEWS.Direct! Volume 02, Issue 03

July - September 2010

ISSN 0975-6043

EDITORIAL - Professor Semra Kahraman

INTERVIEW - Dr Nalini Mahajan FEATURED MINI-REVIEW

INDIAN EDITION

IVF NEWS.Direct! www.ivfnewsdirect.com Editorial Advisory Board

Professor Alan Copperman Director, Division of Reproductive Endocrinology; Vice-chairman, Department of ObGyn and Reproductive Science, Mount Sinai Medical Center; Co-director, Reproductive Medicine Associates of New York, US

CONTENTS FEATURED ARTICLE

REVIEW ARTICLE

Professor Zeev Blumenfeld Associate Professor, Reproductive Endocrinology, Department of ObGyn, Rambam Medical Center, Technion-Faculty of Medicine, Haifa, Israel

Dose-specific effects of r-hCG on obese women undergoing ART Prof Semra Kahraman, Dr Mustecep Kavrut

INTERVIEW

Dr Peter Hollands Senior Lecturer in Biomedical Science, Department of Biosciences, University of Westminster, UK

Primary ovarian insufficiency: Review of literature Dr Shylaja B Rajiv

Clinical perspectives: Primary ovarian insufficiency Dr Lawrence M Nelson

FEATURED MINI-REVIEW

Professor Semra Kahraman

Metformin before and during ART effective in non-obese PCOS women

Director, Istanbul Memorial Hospital, ART and Genetics Center, Istanbul, Turkey

MINI-REVIEWS

Professor Pratap Kumar

ART

Professor and Head, Department of ObGyn; Director, Manipal Assisted Reproduction Centre, Manipal University, India

Dr Luciano Nardo Consultant, St. Maryâ&#x20AC;&#x2122;s Hospital, CMFT University Hospitals, Manchester; UK Honorary Lecturer, University of Manchester; UK Director, North West Fertility, UK

Professor Robert Norman Director, Robinson Institute, Adelaide; Professor, Department of ObGyn, University of Adelaide, Australia

Professor John C Petrozza Chief, Division of Reproductive Medicine & IVF Massachusetts General Hospital, Harvard Medical School, Boston, USA

Professor Gamal Serour Director, International Islamic Center for Population Studies and Research, and Al Azhar ART Unit; Department of ObGyn, Al Azhar University; Clinical Director, The Egyptian IVF & ET Center, Maadi, Egypt

Antisperm antibodies may not adversely influence ART pregnancy rates

FSH administration during hCG trigger may improve oocyte developmental competence in IVF cycles

Japanese study reports lower ectopic pregnancy risk with frozen-thawed single blastocyst transfer

Editorial Team Managing Editor Dr B M John Assistant Editor Dr Shylaja B Rajiv

INFERTILITY

Assistant Copy Editor Amoolya Moses Research Analysts Dhanya Mohan Dr Raghavendra Rao Design Veeresh Mathapati Abhilash A

INFORMATION For contributions, author guidelines, and comments: editor@ivfnewsdirect.com For advertisements and reprints: sales@ivfnewsdirect.com Terms of use: http://www.ivfnewsdirect.com/?page_id=16 Editorial Process: http://www.ivfnewsdirect.com/?page_id=14

Designed and Published

on behalf of IVF NEWS.Direct! by iLogy Healthcare Solutions Disclaimer Views and opinions expressed in this publication are not necessarily those of iLogy. While every effort has been made to ensure accuracy of the information published in this edition, neither iLogy and its employees nor its information vendors accept responsibility for any errors or omissions. Further, iLogy and its information vendors do not take any responsibility for loss or damage incurred or suffered by any reader of this journal as a result of accepting any invitation/offer published in this edition. iLogy reserves the right to use the information published herein in any manner whatsoever. No part of this publication may be reproduced in any form without the written permission of the publisher.

Better pregnancy rates reported with single postovulation IUI compared to single pre-ovulation IUI CRYOPRESERVATION

Study reports effective blastocyst cryopreservation using closed blastocyst vitrification system GENETICS

Study on vertical transmission of hepatitis B infection to offspring through germ cells

NEWS

24 25

ESHRE defines poor response to ovarian stimulation

British IVF data shows association between number of oocytes retrieved and live birth rate

Semen washing does not eliminate risk of HPV infection

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January - March 2012

FEATURED ARTICLE Primary ovarian insufficiency: Review of literature indicate the reversibility of the condition. Recent research has shown that among the different terminologies, POI can include the entire spectrum of impaired ovarian function and corresponds accurately to the clinical scenario; hence, it could be preferred over the others.

Dr Shylaja B Rajiv Assistant Editor, IVF NEWS.Direct! Email: shylaja@ilogy.com

Primary ovarian insufficiency (POI), a condition characterized by hypoestrogenism, elevated serum gonadotropin levels, and amenorrhea, can occur at any age, but is more common in women younger than 40 years.1 As per the American Pregnancy Association (APA), POI affects around 1 in every 1,000 and 1 in every 100 women in the age groups of 15 to 29 years and 30 to 39 years, respectively.2 Although the common etiologies of POI are fragile X premutations, autoimmune disorders, and chromosomal abnormalities, it is idiopathic in most of the cases.3 Resolution of symptoms, bone protection, and psychosocial support form the main management strategies for this devastating condition.3

Etiopathogenesis As per the World Health Organization (WHO), ovarian insufficiency can occur either due to primary disorder in the ovary in which the reproductive organ does not function normally on gonadotropin stimulation, or secondary factors, wherein adequate gonadotropin stimulation is not provided by the hypothalamus and pituitary. According to the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), women with POI can be categorized into two groups:5 •• Follicle depletion: No follicles left and no chance of its further production •• Follicle dysfunction: Follicles may be present but do not function properly

Primary ovarian insufficiency: Is the term apt? Some of the common alternative names of POI include premature ovarian failure (POF), early menopause, and premature menopause. POI may be a more precise name, as it can be altered suitably to depict the state of ovarian function.3 Although many feel that POI is more accurate and informative to the patients than the other suggested names, Shelling in a 2010 review, indicates it to be a vague term.4 Early menopause, although a very broad term that includes all women reaching menopause before 45 years, is also used. Premature menopause implies permanent infertility, but may not be true in all cases as ovulation and pregnancy can sometimes occur at a later duration.4

Several factors have been suggested to be responsible for the occurrence of POI, as enumerated in Table 1.6, 7 Table 1: Etiology of POI Causes Genetic links

•• ••

Literature search demonstrates that there is no clear consensus on the appropriate term to be used for the condition. Analyzing several studies, Shelling in his review recommends the use of POF till international consensus is attained.4 However, POF is an extreme condition characterized by the complete depletion of primordial follicles, having no methods, currently, to assess this absence of follicles in the ovary. Considering this concept, several researchers do not agree on the term premature ovarian failure as it is not yet proven. Furthermore, several studies indicate that POF is a poor definition of the disorder that has a complicated presentation and course. Some scientists prefer to name it premature ovarian dysfunction (POD) in order to avoid the usage of the word ‘failure’ and to www.ivfnewsdirect.com

•• •• •• •• ••

Gonadal dysgenesis

Infection Autoimmune

•• •• •• •• •• •• •• ••

Idiopathic Iatrogenic Enzyme deficiencies

•• •• •• •• •• •• ••

Conditions Familial ovarian failure Fragile X syndrome Small X chromosome defects FSH and LH receptor mutations Blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) Galactosemia Changes in several genes such as BMP15, FOXL2, FOXO3, FMR1, FMR2, INHA, ESR1, ESR2, SF1, and CYP19A1 46, XX gonadal dysgenesis 46, XY gonadal dysgenesis Turner syndrome Perrault syndrome Viral oophoritis Isolated autoimmune ovarian failure Autoimmune polyendocrinopathy syndrome 1 and 2 In conjunction with other autoimmune diseases Causative agents not known Pelvic surgery Chemotherapy Radiotherapy Cholesterol desmolase deficiency 17-alpha-hydroxylase deficiency 17-20-desmolase deficiency

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•• Physical examination, to specifically rule out thyroid or adrenal/estrogen insufficiency (fasting glucose, TSH, and antithyroid antibodies) •• LH, FSH, and estradiol levels, twice, with a gap of six weeks between the tests •• Bone densitometry, if there is significant length of amenorrhea •• Screening for fragile X syndrome •• Adrenocorticotropic hormone stimulation test, if there is suspicion of Addison’s disease (gastrointestinal symptoms, hypotension, weakness, anorexia, and hyperpigmentation) •• Karyotyping for mosaicism with a Y chromosome that may require gonadectomy in women <30 years. Not required in women >30 years since the occurrence of gonadal tumors is very rare

Clinical presentation POI, a continuum of disorders, is categorized into four clinical states (Table 2); the patients’ condition can unpredictably change from one to another, with the resumption of normal ovarian function (for a period of time) in rare cases.8 Table 2: Clinical presentation in the spectrum of POI2 Clinical State

Menses

Fertility

Serum FSH

Occult POI

Regular

Reduced

Normal

Biochemical POI

Regular

Reduced

Raised

Overt POI

Irregular or absent

Reduced

Raised

POF

Absent

Zero

Raised

The clinical presentation varies from the absence of symptoms to having hot flashes, night sweats, irregular periods, change in menstrual flow and duration, irritability, reduced sexual drive, thinning and drying of vagina, and painful sexual intercourse.2 Failure in resumption of menstruation following pregnancy or after cessation of oral contraceptive pills is one of the common presentations of POI.4

Several other studies have suggested the measurement of FSH levels at different times, in order to exclude intermittent ovarian activity from the differential diagnosis of elevated gonadotropins.4 Other etiological factors of amenorrhea, such as polycystic ovarian syndrome, pregnancy, thyroid dysfunction, and hyperprolactinemia, have to be excluded to confirm the diagnosis.4 A 2008 report of the Practice Committee of the American Society for Reproductive Medicine (ASRM) suggests that the persistent measurement of FSH levels in the menopausal range is needed to confirm the disease.10

Spencer Richlin, in his 2006 study, noted that dysfunctional uterine bleeding and oligomenorrhea are generally found in 50% of cases, while 25% present with menstrual cessation post oral contraceptive use or abrupt-onset amenorrhea.9 Patients need to be counseled that the condition is not caused by contraceptives, and that only the symptoms are obscured by it. Furthermore, 4% of the subjects having spontaneous POI may present with adrenal insufficiency, with a subsequent enhanced risk for adrenal crisis. Considering this, it is important to monitor adrenal antibody status prior to oocyte donation along with yearly testing of adrenal antibody levels, to assess new onset adrenal insufficiency.4

A 2002 study by Alzubaidi et al investigated the experiences of 50 spontaneous POI patients with regard to the promptness of their diagnosis and initial presenting symptoms. It was found that the most common initial symptom was change in the menstrual pattern, and diagnosis required >5 years to be established in 25% of women. More than 50% of the patients consulted ≥3 clinicians prior to diagnosis of the disease. Furthermore, there was a perception among women that a more aggressive approach is needed to evaluate secondary amenorrhea and oligomenorrhea.11

Diagnosis The criteria to confirm the diagnosis of POI is not standardized as no internationally reputed organization has defined them.8 A 2006 study states that the condition can be diagnosed based on4 •• presence of amenorrhea for 4 months •• random testing of FSH levels, indicating values >40 mIU/mL in women aged below 40 years

Treatment NICHD states that ovarian function cannot be restored by any proven therapeutic strategy.12 Hence, the primary goal of the treatment is to manage the hypoestrogenic state linked to POI and prevent its long-term adverse sequelae. The management of POI involves addressing both the physical and emotional well-being of women.8

The study also proposed the following investigations for diagnosing POI:4 •• History, particularly age (<40 years), amenorrhea/ oligomenorrhea, and hot flashes www.ivfnewsdirect.com

January - March 2012

Hormone replacement therapy (HRT) Literature search has shown that women with POI may require long-term hormone replacement therapy (HRT), 2

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either with estrogen or in combination with progestin, in order to prevent osteoporosis and symptoms of estrogen deficiency. It has been reported that young women may particularly need HRT for close to 40 years.8, 13 A 2008 study published in Clinical Endocrinology indicates that although there is lack of sufficient evidence (randomized controlled studies) on the benefits of combined HRT, many specialists agree on its usage in young patients till menopausal age.8

women aged ≥50 years.15 It has been observed that many women benefit from HRT, but the long-term exposure to hormones may enhance the baseline risk for secondary malignancies, including breast cancer. Considering this, the author emphasizes that HRT should not be reflexively administered to all childhood cancer survivors with POI and stresses on the importance of a thorough and balanced discussion with patients on the advantages and limitations before initiating the treatment.

Various hormonal regimens indicated for the treatment of POI are given below. •• Estrogen is administered either orally or transdermally, continuously or cyclically (daily for 21 days and resumed after a 7-day gap). •• Progestins are cyclically administered 10-14 days every month in order to avert endometrial hyperplasia (due to unopposed estrogen). The recommended protocols are 200 mg daily of micronized progesterone or 10 mg of medroxyprogesterone for 10-12 days every month. •• Androgens may have to be cautiously prescribed in women experiencing persistent fatigue, poor wellbeing, and low libido, in spite of sufficient estrogen replacement, and in cases where depression is not the cause or is already treated.

According to a recent review by Gelbaya et al (2011), there is a lack of consensus on the optimal HRT regimen for maximizing the reproductive potential in both young and old groups of POI patients, although the dose and duration of the treatment is generally based on the patient’s height and Tanner stage of development (in prepubertal individuals).16

Maintaining bone health Several trials have confirmed the adverse impact of POI on bone mineral density. In one such controlled crosssectional study, Amarante et al (2011) demonstrated that patients with primary ovarian insufficiency have significantly reduced bone mineral density when compared to age-matched premenopausal women. Furthermore, such women have higher prevalence of osteopenia or osteoporosis in comparison to those following natural menopause. In light of the findings, the investigators emphasized the need for early initiation of medical treatment for women with POI to maintain the existing bone mass.17

A 2009 review by Nelson noted that there is no trial directly comparing the numerous hormonal therapies for women with POI. The transdermal administration of 100 μg of estradiol daily is effective in reducing the symptoms since it helps in achieving average levels of estradiol (around 100 pg/mL), as found in those with regular menstrual cycles.8 Previously, a 2006 study by Richlin reported the benefits of 100 µg of estradiol patch in most of the patients, and administration of 10 mg/ day of cyclic medroxyprogesterone acetate for 12 days to produce regular withdrawal bleeding.9

In a 2010, open-label, randomized controlled, crossover study, Crofton et al found that treatment for over 12 months with 100 μg transdermal estradiol, daily for week 1, followed by 150 μg of estradiol for weeks 2-4, and 200 mg of vaginal progesterone twice daily for weeks 3-4 was effective in young women with POI. The regimen was beneficial in terms of acquisition of bone mass on the lumbar spine that is facilitated by enhanced bone formation and reduced bone resorption.18

Reiterating the benefits of HRT, Hershlag et al (2011) indicated that the immediate concerns in iatrogenic POI, especially among teenage girls surviving cancer, are its adverse effects on the physical, psychosocial, and sexual development, and ability to achieve adulthood. The researchers support the use of meticulous, graduated HRT in adolescents in order to mimic Tanner stages of pubertal development, thereby aiding them to smoothly graduate into adulthood.14

A 2009 review by Nelson highlighted the significance of measuring bone mineral density to identify the baseline values in order to educate women on the strategies needed to maintain bone health.8 However, there is a lack of sufficient evidence on the daily requirements of calcium and vitamin D, and the recommended intensity and frequency of weight-bearing exercise in women with POI. Considering this, the researcher suggests that women with POI can follow the recommended dosages proposed by the North American Menopause Society (NAMS) for

Another 2011 review notes that information on the long-term safety of HRT in survivors of childhood cancer is scarce, although several trials have evaluated the benefits and risks of exogenous hormones in menopausal www.ivfnewsdirect.com

January - March 2012

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January - March 2012

perimenopausal and postmenopausal women: serum 25-hydroxyvitamin D level ≥30 ng/mL (75 nmol/L) and 1,200 mg of elemental calcium daily.8, 19 In addition to this, women should engage themselves in physical activities, such as walking, jogging, climbing stairs, and other resistance exercises, to decrease the risk of osteoporosis.

•• Offer accurate information sources related to the diagnosis/condition itself for better understanding •• Explain the possibility of intermittent and unpredictable ovarian function that could very rarely result in spontaneous pregnancy years after the diagnosis

Similarly, the National Osteoporosis Foundation (NOF) suggests that no treatment can completely prevent bone loss, but can only aid in slowing the process, and recommends the following in women at high risk of osteoporosis:11 •• Minimum of 1,200 mg of calcium as dietary intake per day, if not, calcium supplements •• Daily intake of 400 IU to 800 IU of vitamin D to facilitate calcium absorption •• Regular weight-bearing and muscle-building physical activity to build bone and muscle strength, and enhance flexibility •• Maintain adequate estrogen levels, as it helps to prevent bone loss and osteoporosis

ART Infertility occurs commonly in patients with POI, with no proven therapeutic strategy for restoring ovarian function.5 Although spontaneous conception can occur in 5-10% of women with POI, majority may require assisted reproductive technology.5 In young cancer patients with a high risk of iatrogenic infertility, cryopreservation of embryos, ovarian tissues, and oocytes, and in vitro maturation of oocytes hold promise in preserving future fertility.24 Studies have noted that medical therapies do not increase the likelihood of spontaneous conception in women with an almost definite diagnosis of POI. Conventional ovulation induction with or without gonadotropin suppression may not enhance the fertility rates. Some uncontrolled trials indicate that fertility rates in women with POI due to autoimmune etiology may improve with glucocorticoid treatment. However, it cannot be used in clinical settings owing to the lack of good autoimmune markers for ovarian failure as well as sufficient evidence on the risks associated with the treatment.6

Emotional health The diagnosis of POI is difficult and emotionally traumatic to women.20 In a 2009 cross-sectional study, Orshan et al demonstrated a substantial reduction in scores on the self-esteem and perceived social support scale in women with POI (hypergonadotropic hypogonadism), when compared to controls.21 The researchers indicated the need to develop strategies to decrease the emotional suffering of such women by improving their self-esteem and social support.

The introduction of oocyte and embryo donation over the past decade has provided hope to many women with POI.6 A 2011 review has proposed ART with donated gametes as the only realistic strategy for women with POI to achieve motherhood. The outcomes of ART cycles are based on uterine and endometrial development, which in turn is significantly affected by HRT modality and age at which it is initiated.16 Similarly, several studies, including two reviews (Richlin; Rebar et al), have indicated that oocyte donation is still the best strategy to achieve pregnancy in such patients.13, 25 Reiterating the benefits of oocyte donation, a retrospective analysis in 2009 demonstrated its success in women with POI and physiological menopause.26

A 2005 observational study noted that the emotional distress levels varied by the way the clinicians informed the patients about their health condition. To better face the situation, the patients want their clinicians to spend more time explaining and educating them about POI.20 A cross-sectional study by Ventura et al found a significant association between spiritual and functional well-being scores. It was observed that methods to improve spiritual well-being, in areas such as purpose, meaning, and inner peace, may aid in decreasing the emotional suffering.22 A healthcare provider plays a vital role in helping women with POI cope with the condition. Several studies have suggested steps to help such women after the diagnosis, some of which are listed below.23 •• Provide emotional support and confidence •• Encourage patients to freely express their feelings •• Persuade patients to contact support groups to decrease the feelings of loneliness and isolation www.ivfnewsdirect.com

Claimed to be the first study reporting the birth of twins using sister-donor oocytes in Ireland, Sills et al indicated that women with POI can conceive from oocyte donation IVF cycles. The case study involved the use of IVF on a POI patient using the oocytes donated by her non-identical (dizygotic) twin sister. The study findings also underscored the key role of pretreatment counseling in sister oocyte 4

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donation similar to any other kind of donor gamete therapy.27

conception can occur in some women (including those taking exogenous estrogen with or without progestogen) since ovarian follicles may remain in some cases.

International statements Various internationally reputed organizations have issued statements regarding the diagnosis and treatment of POI; some of them have been briefed below.

The European Menopause and Andropause Society (EMAS), in 2010, issued a position statement on POI management.28 •• Diagnosis is confirmed when the levels of FSH are >40 IU/L and estradiol <50 pmol/L, in cases where there is no bilateral oophorectomy. •• Patients also need to undergo autoimmune screening for polyendocrinopathy, thyroid function tests, karyotyping (if <30 years), and bone mineral density evaluation. •• Patients have to be educated on the condition and provided enough information to cope with the diagnosis. •• Early ovarian failure, if left untreated, enhances the risk of several conditions such as osteoporosis, dementia, cardiovascular disease, and cognitive decline and parkinsonism. •• Hormone treatment is the mainstay of managing POI, which has to be continued until natural menopause. •• Although spontaneous ovulation may occur rarely, the best chance of achieving pregnancy is by oocyte donation IVF cycles. •• Women with POI will need treatment in various areas, including general health, emotional health, bone health, and reproductive health; hence, a multidisciplinary team approach is required for optimal management.

The 2008 report of the Practice Committee of ASRM indicates the following with respect to POI:9 •• Premature menopause occurs in around 16% of women, who are carriers of fragile X syndrome premutation. •• POI may occur in 10% to 60% of patients with Addison’s disease having autoimmune lymphocytic oophoritis, which is very rare and occurs in 1 among 1 million women. •• Autoimmune abnormalities, especially autoimmune thyroiditis, may occur in up to 40% of women with POI. •• A slight increase in the prevalence of POI is noted in women with insulin-dependent diabetes mellitus, parathyroid disease, and myasthenia gravis, when compared to healthy women. •• Detailed family history has to be procured since many autosomal disorders, such as mutations in the FSH receptor gene, galactose-1-phosphate uridyltransferase (GALT) gene, chromosome 3q containing blepharophimosis gene, autoimmune regulator (AIRE) gene, and phosphomannomutase 2 (PMM2) genes, are linked to ovarian failure. •• Although ovarian biopsy is not suggested in routine clinical practice, women should be screened for other indications by measuring thyroid autoantibodies, TSH, fasting glucose, and electrolytes to rule out autoimmune POI as a part of polyglandular syndrome. •• Currently, there is lack of a validated serum antibody marker that can confirm the diagnosis of autoimmune POI. •• Karyotyping should be obtained in women <30 years to exclude short arm deletion, sex chromosome translocation, or occult Y chromosome that is linked to the enhanced risk of gonadal tumors. •• No treatment is found to be effective in infertile patients with autoimmune POI, as per a prospective controlled study. •• Estrogen and progestin treatment should be administered to POI patients in order to stimulate and maintain secondary sexual characteristics and decrease the risk of osteoporosis development. •• In rare instances, spontaneous ovulation and www.ivfnewsdirect.com

January - March 2012

Implications for future research Although no evidence exists to indicate the increasing prevalence of POI, the condition is gaining more importance, considering that women want to delay motherhood to a later age.4 Furthermore, in most cases, the diagnosis of POI is known to cause adverse psychological impact, but there is underestimation of the emotional importance of the disorder. Hence, women at risk of POI need to be educated and counseled on the risk of delaying motherhood and methods to restrict the detrimental effects of early menopause, along with providing emotional support to overcome the shock of the life-altering diagnosis.4 In a 2011 literature review and multi-disciplinary scientific workshop focusing on POI consensus building, Cooper et al stressed on the lack of sufficient knowledge on several aspects of the condition, including etiology, psychosocial impact, natural history, and medical and psychosocial 5

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treatment. This calls for an international research consortium and disease registry, under the review and guidance of a reputed organization, to serve as a pathway for comprehensively enhancing the basic and clinical knowledge about POI. This could also aid in providing clinical samples and data, which in turn could facilitate defining specific pathogenic mechanisms. Furthermore, Cooper et al opined that an international collaborative approach combining the patient registry structure and principles of integrative care and community-based participatory investigation would help in advancing our understanding of primary ovarian insufficiency.29

January - March 2012

adolescent cancer survivors should be treated. J Pediatr Adolesc Gynecol. 2011 Apr;24(2):101-3. 15. Fish JD. Part 1: Hormone replacement for survivors of childhood cancer with ovarian failure--when is it worth the risk? J Pediatr Adolesc Gynecol. 2011 Apr;24(2):98-101. 16. Gelbaya T, Vitthala S, Nardo L, Seif M. Optimizing hormone therapy for future reproductive performance in women with premature ovarian failure. Gynecol Endocrinol. 2011 Jan;27(1):1-7. 17. Amarante F, Vilodre LC, Maturana MA, Spritzer PM. Women with primary ovarian insufficiency have lower bone mineral density. Braz J Med Biol Res. 2011 Jan;44(1):78-83. 18. Crofton PM, Evans N, Bath LE, et al. Physiological versus standard sex steroid replacement in young women with premature ovarian

References

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19. North American Menopause Society. The role of calcium in peri-

02. Premature Ovarian Failure: Premature Menopause. American

and postmenopausal women: 2006 position statement of the

Pregnancy Association. Available at http://www.americanpregnancy.

North American Menopause Society. Menopause. 2006 Nov-

org/womenshealth/pof.htm. Accessed October 28, 2011.

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03. Welt CK. Primary ovarian insufficiency: a more accurate term for

20. Groff AA, Covington SN, Halverson LR, et al. Assessing the

premature ovarian failure. Clin Endocrinol (Oxf). 2008 Apr;68(4):499-509.

emotional needs of women with spontaneous premature ovarian

04. Shelling AN. Premature ovarian failure. Reproduction. 2010

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21. Orshan SA, Ventura JL, Covington SN, Vanderhoof VH, Troendle

05. Premature Ovarian Failure. Eunice Kennedy Shriver National

JF, Nelson LM. Women with spontaneous 46,XX primary ovarian

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insufficiency (hypergonadotropic hypogonadism) have lower

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06. Premature Ovarian Failure. endocrineonline.org. Available at http://www.endocrineonline.org/pdf%20box/pof.pdf.

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23. Nelson LM. Spontaneous Premature Ovarian Failure: Young Women,

Arch Gynecol Obstet. 2011 Mar;283(3):635-43.

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08. Nelson LM. Clinical practice. Primary Ovarian Insufficiency. N Engl J

24. Goswami D, Conway GS. Premature ovarian failure. Hum Reprod

Med. 2009 Feb 5;360(6):606-14.

Update. 2005 Jul-Aug;11(4):391-410.

09. Richlin S. Premature Ovarian Failure. In: 2006 Art Resource

25. Rebar RW. Premature ovarian failure. Obstet Gynecol. 2009

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10. Practice Committee of American Society for Reproductive

26. Ameratunga D, Weston G, Osianlis T, Catt J, Vollenhoven B. In vitro

Medicine. Current evaluation of amenorrhea. Fertil Steril. 2008

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are there differences in pregnancy outcomes in women with

11. Alzubaidi NH, Chapin HL, Vanderhoof VH, Calis KA, Nelson LM.

premature ovarian failure (POF) compared with women with

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28. Vujovic S, Brincat M, Erel T, Gambacciani M, et al. EMAS position

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REVIEW ARTICLE Dose-specific effects of r-hCG on obese women undergoing ART batch-to-batch variation in activity, which may influence the clinical outcomes. Furthermore, low purity of urinary preparations prevents use of the SC route, making IM administration as the only option.

Prof Semra Kahraman, Dr Mustecep Kavrut IVF and Reproductive Genetics Unit, Istanbul Memorial Hospital, Istanbul, Turkey E-mail: semkahraman@gmail.com

Introduction Obesity is one of the major health problems today due to its dramatically increasing frequency. Besides the common metabolic and cardiovascular risks, the potential negative effects on fertility are now being discussed more extensively.1, 2 High BMI may have a vital impact on the metabolism and distribution of human chorionic gonadotropin (hCG), reducing its bioavailability, and possibly decreasing final oocyte maturation, thus negatively affecting the success of in vitro fertilization (IVF).3-9

More recently, recombinant hCG (r-hCG) is being used in routine clinics, with major advantages such as efficacy on final follicular maturation, good tolerability, and the option of both SC and self administration.8, 14-16 Thus, patients can self-administer drugs throughout the treatment without the need for IM.16

Being overweight or obese may reduce a woman’s fertility and increase the risks associated with pregnancy; however, its impact on IVF cycle outcomes is controversial. Several studies have reported that although there is no negative relation between BMI increase and IVF success rates, the cancellation rates are higher. In addition, studies indicating no adverse effect of obesity on IVF therapy agree on the necessity of high-dose gonadotropin administration.10-12

Relation between BMI and hCG dosage The optimum dose of r-hCG necessary for the final follicular maturation is commonly accepted to be 250 µg.19 However, Chan et al compared two groups administered with 250 µg and 500 µg of r-hCG, and obtained similar results in both the subsets.20 In contrary, other studies have stressed on the need for increasing hCG dosage due to diminished pregnancy rates in obese patients undergoing assisted reproduction.10-11, 21-22 Adiposity may possibly affect the intrafollicular microenvironment and thus oocyte maturation. The bio-availability of hCG and BMI are reported to be negatively correlated.23

It is observed that the absorption of ovarian stimulation drugs may be reduced with subcutaneous (SC) administration due to the increased SC adipose tissue in obese women. However, a study comparing intramuscular (IM) vs. SC routes for administration of recombinant follicle stimulating hormone (rFSH) in high BMI women did not show any statistically significant differences between the routes of administration in terms of drug pharmacokinetics.13

Administering three different doses of u-hCG, Detti et al (2007) reported that (i) BMI may be used as a predictor of hCG concentration (ii) hCG dose titration should be carried out with respect to BMI.24 Moreover, the negative correlation between BMI and serum hCG levels were reported as being independent of the administration procedure (IM or SC).25 However, other studies have been unable to find any negative impact of obesity on ART outcomes.

Role of hCG in oocyte maturation The administration of hCG helps in inducing final follicular maturation and rupture. When sufficient amount of hCG is present in the ovarian follicles, oocyte reaches the metaphase II stage and gains fertilization ability. The success of mature oocyte retrieval is dependent on the critical serum hCG level for meiosis initiation and release of the cumulus-oocyte complex into the follicular fluid (FF). To date, the hCG used in IVF cycles is extracted from the urine (u-hCG) of postmenopausal women. However, the major disadvantages of this mode of extraction are unreliable source, lack of purity, and

A common conclusion of several studies is that obese patients require more gonadotropin.10-12 Stefanis et al measured serum hCG concentrations in patients undergoing IVF treatment after 12 or 36 hours of SC injection of 5,000 IU hCG. No correlation was found between serum levels and the number of retrieved or fertilized oocytes. Moreover, no link was established between BMI and hCG levels.26 Another study by Orvieto et al compared obese (BMI≥30) and non-obese (BMI<30) groups undergoing IVF and noted that obese women require a longer stimulation and a higher dose of gonadotropin; nevertheless their pregnancy rates were lower.27

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Urinary and r-hCG dosages in high BMI In a prospective randomized study comparing 250 µg r-hCG and 5,000 IU u-hCG, the researchers found no difference in the number of oocytes, clinical and ongoing pregnancies, and rates of delivery and miscarriage between the two groups. At the end of this study, it was concluded that the efficacy of 250 µg r-hCG and 5,000 IU u-hCG were similar.14 A study by Carrell et al, which compared two groups of patients: BMI>30 kg/m2 and <30 kg/m2, found an inverse correlation between BMI and intrafollicular hCG concentration. Intrafollicular hCG was also significantly lower in the >30 BMI group. This in turn, may be related to a concurrent decrease in embryo quality, and pregnancy rates in obese and overweight patients.17

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In this study, when we categorized the cases into two subgroups according to BMI (26-30 kg/m2 [mean=28 kg/ m2] vs. >30 kg/m2 [mean=33 kg/m2]), we observed that there were no differences in clinical outcomes, mainly MII oocytes. The study did not involve patients with polycystic ovarian syndrome. BMI was assessed as an obesity marker. Although the hCG values in serum and follicular fluid were higher in the 500 µg r-hCG group compared to those who received 250 µg r-hCG, the total and mature oocyte rates and pregnancy results were similar. No difference in ovarian hyperstimulation syndrome (OHSS) rates was observed between total and categorized BMI groups. Achieving pregnancies in high BMI patients with even low-hCG levels in the blood and follicular fluid connotes that the given dose of r-hCG was above the mean value of the minimum threshold required for follicular maturation. Based on the results of our study, we concluded that 250 µg r-hCG is sufficient for obtaining final follicular maturation in patients with a BMI>26 kg/m2. Using higher doses of hCG increases the cost, but does not provide any additional clinical benefit in obese patients.

However, there is limited data in literature comparing the effectiveness of 500 µg over 250 µg of r-hCG in obese ART patients. In a prospective randomized study conducted by our group between December 2007 and December 2008, doses of 250 µg and 500 µg r-hCG were compared in IVF patients with a BMI≥26 kg/m2.18 We administered 54 and 51 patients with 250 µg and 500 µg r-hCG, respectively, and concluded that the outcome parameters were not affected by the dose and 250 µg r-hCG was as efficient as 500 µg r-hCG.

In light of these findings, we have conducted another trial to investigate the efficacy of 250 µg r-hCG in patients with a higher BMI (≥30) and compare the two doses of r-hCG in obese (BMI≥30 kg/m2) and highly obese Table 1: Comparison of demographic and IVF outcomes between two doses of r-hCG (BMI≥35 kg/m2) women undergoing IVF. in women with high BMI18 The study is currently under review for Parameters Evaluated 250 µg 500 µg P publication. In the first (previous) study Age (years) 31.2±4.3 32.2±4.3 0.120a on patients with BMI≥30, regardless of BMI 29.7±3.1 29.4±2.9 0.599b the dose of r-hCG (250 µg or 500 µg) No. of cycles 2.3±1.9 2.8±2.2 0.167b injected for final maturation of follicles, no difference was observed in the Baseline FSH (IU/L) 6.1±2.5 7.5±4.1 0.380a a obtained total or MII oocyte and cycle No. of days of stimulation 9.2±1.4 9.4±1.2 0.326 parameters. In our second trial, we have Total dose injected 3093.8±1335.4 3157.6±1319.5 0.569 a demonstrated that successful results No. of oocytes 13.2±5.2 12.6±5.2 0.583 may also be obtained with 250 µg r-hCG b No. of MII oocytes 10.3±4 9.6±4.5 0.290 in highly obese women. MII/total oocytes 0.8±0.15 0.78±0.18 0.772b No. of fertilized oocytes

7.8±3.7

7±3.8

0.173b

No. of transferred embryos

2.4±0.7

2.5±0.7

0.522b

73.9±18.8

146±48.1

<0.001a

16±12.4

35.9±25.5

<0.001b

FF:serum hCG ratio

0.21±0.17

0.24±0.13

0.118b

Pregnancy rate (%)

57.4

54.9

0.796c

Clinical pregnancy rate (%)

50.0

47.1

0.763c

0.60±0.27

0.60±0.26

0.937a

14.8

20.8

0.574c

2 (3.7%)

1 (1.9%)

Serum hCG levels on OPU day (mIU/mL) FF hCG levels on OPU day (mIU/mL)

Implantation rate Spontaneous abortion rate (%) OHSS Note: Data are presented as mean±SD FF=follicular fluid a P values for student T test

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P values for Mann-Whitney U test P values for x2 test

b c

As a conclusion, we have shown for the first time that 250 µg of r-hCG is sufficient and safe to trigger ovulation in women with BMI of ≥30 kg/m2. No clinical or statistical advantage could be demonstrated for the higher dose of r-hCG in obese and highly obese patients. Since low doses of r-hCG give similar results, additional doses are unnecessary. However, increasing the number of obese patients enrolled in well-designed, additional studies will augment the statistical power, allowing us to draw a conclusion. COPYRIGHT © 2012

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January - March 2012

References

gonadotropin (rhCG) in assisted reproductive technology: results

01. Metwally M, Li TC, Ledger WL. The impact of obesity on female

of a clinical trial comparing two doses of rhCG (Ovidrel) to urinary

reproductive function. Obes Rev. 2007 Nov;8(6):515-23.

hCG (Profasi) for induction of final follicular maturation in in vitro

02. Obesity and overweight. World Health Organization. Available at

fertilization-embryo transfer. Fertil Steril. 2001 Jul;76(1):67-74.

http://www.who.int/mediacentre/factsheets/fs311/en/. Updated

17. Carrell DT, Jones KP, Peterson CM, Aoki V, Emery BR, Campbell

March, 2011. Accessed May 03, 2011.

BR. Body mass index is inversely related to intrafollicular HCG

03. Bohler H Jr, Mokshagundam S, Winters SJ. Adipose tissue and

concentrations, embryo quality and IVF outcome. Reprod Biomed

reproduction in women. Fertil Steril. 2010 Aug;94(3):795-825.

Online. 2001;3(2):109-111.

04. Bstandig B, Schumaker C, Isnard V, Isnard V, Ferrari P, Bongain

18. Kahraman S, Karlikaya G, Kavrut M, Karagozoglu H. A prospective,

A. Influence of Body Mass Index (BMI) on Successful Ovulation

randomized, controlled study to compare two doses of

Triggering by Urinary hCG (u-hCG) Versus Recombinant hCG

recombinant human chorionic gonadotropin in serum and

(r-hCG). Fertil Steril. 2005 Sep;84(Suppl 1):S422-423.

follicular fluid in woman with high body mass index. Fertil Steril.

05. Harlass FE, Plymate SR, Fariss BL, Belts RP. Weight loss is associated

2010 Apr;93(6):2084-7.

with correction of gonadotropin and sex steroid abnormalities in

19. Al-Inany H, Aboulghar MA, Mansour RT, Proctor M. Recombinant

the obese anovulatory female. Fertil Steril. 1984 Oct;42(4):649-

versus urinary gonadotrophins for triggering ovulation in assisted

652.

conception. Hum Reprod. 2005 Aug;20(8):2061-73.

06. Hollmann M, Runnebaum B, Gerhard I. Effects of weight loss on

20. Chan CC, Ng EH, Tang OS, Yeung WS, Lau EY, Ho PC. A prospective,

the hormonal profile in obese, infertile women. Hum Reprod. 1996

randomized, double-blind study to compare two doses of

Sep;11(9):1884-91.

recombinant human chorionic gonadotropin in inducing final

07. Rachoń D, Teede H. Ovarian function and obesity-interrelationship,

oocyte maturity and the hormonal profile during the luteal phase.

impact on women’s reproductive lifespan and treatment options.

J Clin Endocrinol Metab. 2005 Jul;90(7):3933-8.

Mol Cell Endocrinol. 2010 Mar 25;316(2):172-9.

21. Wang JX, Davies M, Norman RJ. Body mass and probability of

08. Salha O, Dada T, Sharma V. Influence of body mass index and self-

pregnancy during assisted reproduction treatment: retrospective

administration of hCG on the outcome of IVF cycles: a prospective

study. BMJ. 2000 Nov 25;321(7272):1320-1.

cohort study. Hum Fertil (Camb). 2001;4(1):37-42.

22. Fedorcsák P, Dale PO, Storeng R, et al. Impact of overweight and

09. Wilkes S, Murdoch A. Obesity and female fertility: a primary care

underweight on assisted reproduction treatment. Hum Reprod.

perspective. J Fam Plann Reprod Health Care. 2009 Jul;35(3):181-5.

2004 Nov;19(11):2523-8.

10. Dechaud H, Anahory T, Reyftmann L, Loup V, Hamamah S, Hedon

23. Dumesic DA, Lesnick TG, Abbott DH. Increased adiposity enhances

B. Obesity does not adversely affect results in patients who are

intrafollicular estradiol levels in normoandrogenic ovulatory

undergoing in vitro fertilization and embryo transfer. Eur J Obstet

women receiving gonadotropin-releasing hormone analog/

Gynecol Reprod Biol. 2006 Jul;127(1):88-93.

recombinant human follicle-stimulating hormone therapy for in

11. Esinler I, Bozdag G, Yarali H. Impact of isolated obesity on ICSI

vitro fertilization. J Clin Endocrinol Metab. 2007 Apr;92(4):1438-41.

outcome. Reprod BioMed Online. 2008 Oct;17(4):583-7.

24. Detti L, Mitwally MF, Rode A, et al. Serum human chorionic

12. Lashen H, Ledger W, Bernal AL, Barlow D. Extremes of body mass

gonadotropin level after ovulation triggering is influenced by the

do not adversely affect the outcome of superovulation and in-vitro

patient’s body mass index and the number of larger follicles. Fertil

fertilization. Hum Reprod. 1999 Mar;14(3):712-5.

Steril. 2007 Jul;88(1):152-5.

13. Steinkampf MP, Hammond KR, Nichols JE, Slayden SH. Effect of

obesity

recombinant

follicle-stimulating

25. Elkind-Hirsch KE, Bello S, Esparcia L, Phillips K, Sheiko A, McNichol

hormone

M. Serum human chorionic gonadotropin levels are correlated with

absorption:subcutaneous versus intramuscular administration.

body mass index rather than route of administration in women

Fertil Steril. 2003 Jul;80(1):99-102.

undergoing in vitro fertilization--embryo transfer using human

14. Driscoll GL, Tyler JP, Hangan JT, Fisher PR, Birdsall MA, Knight DC. A

menopausal gonadotropin and intracytoplasmic sperm injection.

prospective, randomized, controlled, double-blind, double-dummy

Fertil Steril. 2001 Apr;75(4):700-4.

comparison of recombinant and urinary HCG for inducing oocyte

26. Stefanis P, Das S, Barsoum-Derias E, Kingsland C, Lewis-Jones

maturation and follicular luteinization in ovarian stimulation. Hum

I, Gazvani R. Relationship between serum human chorionic

Reprod. 2000 Jun;15(6):1305-10.

gonadotrophin levels and body mass index in women undergoing

15. Kovacs P, Kovats T, Bernard A, Zadori J, Szmatona G, Kaali SG.

in vitro fertilisation cycles. Eur J Obstet Gynecol Reprod Biol. 2007

Comparison of serum and follicular fluid hormone levels with

Jun;132(2):204-8.

recombinant and urinary human chorionic gonadotropin during in

27. Orvieto R, Meltcer S, Nahum R, Rabinson J, Anteby EY, Ashkenazi J.

vitro fertilization. Fertil Steril. 2008 Dec;90(6):2133-7.

The influence of body mass index on in vitro fertilization outcome.

16. Chang P, Kenley S, Burns T, et al. Recombinant human chorionic

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Int J Gynaecol Obstet. 2009 Jan;104(1):53-5.

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January - March 2012

INTERVIEW Clinical perspectives: Primary ovarian insufficiency

Q: A potentially life-altering condition, how do you help your patients cope with primary ovarian insufficiency? Do you think that its prevalence is increasing? A: Primary ovarian insufficiency (POI) is indeed a life-altering condition, with many patients considering the diagnosis as a threat to their identity as a woman. Additionally, the disorder is much more than infertility, involving emotional health, physical health, and spiritual wellness. Our research team takes an integrated approach to this situation. We have learned that we need to know the patient at a personal level in order to care for them appropriately. Before we evaluate them, all of our patients answer a series of questions in writing about themselves and how POI has affected their lives. Dr Lawrence M Nelson Head, Scientific Integrative Reproductive Medicine Group, Intramural Research Program on Reproductive and Adult Endocrinology, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US

I share these writings, called ‘The Patient Narrative’ in confidence with our team. This practice has changed our team dynamics and given greater meaning to our work. The occupational therapy assessment of these women has become a central component of the evaluation, with experts helping people redesign their life in the face of life-altering diagnoses. Our published evidence supports the view that those women who find new meaning and purpose in life, in relation to the diagnosis, seem to do the best.

Email: nelsonl@cc1.nichd.nih.gov

I am not aware of any published evidence that indicates an increase in the prevalence of overt POI; however, as more women delay childbearing into their 30s, the impact of this disorder on society certainly has increased. Dr Nelson, a Commissioned Officer in the U.S. Public Health Service, specializes in women’s health research and leads a research effort on primary ovarian insufficiency. He currently works as a physician and research scientist at NIH in Maryland, USA. Dr Nelson has received board certification from the American Board of Obstetrics and Gynecology for specialization in Reproductive Endocrinology. In addition to earning his MD, he holds an MBA from George Mason University in Virginia, where he concentrated his studies on human resource management and the management of nonprofit organizations. After several years of private practice, he returned to academia to complete a clinical research attachment at Hammersmith Hospital in London, a fellowship in reproductive endocrinology at George Washington University, and a research fellowship at the NIH.

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Q: As a continuum of disorders with several different terminologies, which term would aptly describe the condition in your opinion, and why? A: In my view, Fuller Albright had it right in 1942 when he first named this condition primary ovarian insufficiency. The symptoms may be the same as those seen in women with menopause, but the pathophysiology differs dramatically as menopause results from a depletion of functional primordial follicles. The other commonly used terms such as, ‘premature menopause’ and ‘premature ovarian failure’ are scientifically inaccurate because most women with this condition still have potentially functional follicles remaining in the ovary (about 75% in our most recent publication). Several reports have validated these early findings. Furthermore, the terms menopause and premature ovarian failure are stigmatizing. In a survey, we found that majority of our patients prefer POI to the other terms. The word ‘primary’ refers to the fact that the ovary is the primary 10

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problem and ‘insufficiency’ implies that the condition is a continuum of impaired ovarian function, including diminished ovarian reserve and low responders. Ovarian insufficiency can be occult because the menstrual cycles are still regular, and in such cases, we prefer to use the term ‘occult primary ovarian insufficiency.’ An elevated basal FSH or a low response to gonadotropin stimulation is required to identify women with occult POI.

Q: What are the different therapeutic strategies for POI? In your experience, which is the most useful method? A: As noted above, women with POI need an integrated approach to their care (as a team approach works best), covering issues related to physical health, emotional health, and meaning in life. Q: Since the long-term safety of hormone replacement therapy is not clearly known, would you still recommend it to all your patients? Are there alternative treatment strategies that you prefer to customize for your patients?

Q: What are the tests conducted or the criteria to confirm the disease? Are there any specific guidelines followed in clinical practice? A: To make the diagnosis of overt POI, one has to take oligo/amenorrhea seriously as a marker of something amiss. Girls who have not had their first menses by age 15 and women below 40 years having more than 90 days between menses need evaluation. It is important to first rule out pregnancy, and then measure FSH and prolactin. If FSH is in the menopausal range, the test needs to be repeated a month later. Two FSH levels in the menopausal range, combined with 4 months of irregular menses, makes the diagnosis of overt POI. To determine the mechanism, the following tests can be performed after appropriately informing the patient of the implications of the tests and obtaining their consent: •• Karyotyping that counts 30 cells, for identifying women with Turner syndrome •• Adrenal antibodies measurement, for detecting women with steroidogenic cell autoimmunity (autoimmune oophoritis) •• FMR1 gene assessment for the number of CGG repeats, to identify women with FMR1 premutation

A: By definition, women with POI are less than 40 years old. They are not menopausal women who are young, but are young women who are missing the ovarian hormones that their peers have normally. For such women, it truly is hormone replacement. My first line recommendation is that women with POI do best by replacing the estradiol they are missing through a transdermal or transvaginal delivery of 100 micrograms per day of estradiol. The 100 microgram dose is a full replacement dose of estradiol for young women. These routes of administration avoid the first pass effect on the liver that takes place with oral estrogens. This first pass effect significantly increases the risk of thromboembolic phenomena. Patients need to take a full replacement dose of progestin to reduce the risk of endometrial hyperplasia and endometrial cancer. Available data supports the use of medroxyprogesterone acetate as a first line therapy in a dose of 10 mg/day, for the first 12 days of each month. Patients should keep a menstrual calendar and get a pregnancy test if they fail to menstruate when expected. I recommend that women with POI take this regimen until age 50, which is the average age of menopause.

Q: Several studies have noted the association between POI and FMR1 gene premutation. Considering this, are genetic tests done routinely? What are the implications of the carrier status on patient’s health?

There is limited evidence on other regimens in this population, and I have little confidence in alternative strategies with regard to hormone replacement in this population.

A: There is consensus that all women with POI should be offered testing for the FMR1 gene premutation. Women with the premutation are at risk of having a child with fragile X syndrome, if they are among the 5%-10% of women who get pregnant after the diagnosis of POI. This syndrome is the most common form of heritable mental retardation. The discovery of carrier status has significant implications for other family members with regard to having children with mental retardation. There are also reports that patients who carry the premutation are at risk of developing a neurodegenerative disorder in later life, referred to as fragile X-associated tremor ataxia syndrome (FXTAS). This is more common in men than women. www.ivfnewsdirect.com

Q: Infertility is common in POI patients, having no proven therapeutic strategy for restoring ovarian function. In your experience, what are the interventions that have the likelihood of increasing the chances of conception? Would you say spontaneous conception is possible in such patients? A: There are no proven strategies for increasing the chances of conception by improving ovarian function. It is important to inform patients with POI that there is a 11

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5%-10% chance of spontaneous conception despite the diagnosis. If they are interested in pregnancy, they should do what it takes to assure that there will be sperm waiting for the egg that might ovulate now and then. Sperm can live in a woman’s genital system for 3 to 4 days, so intercourse 2 or 3 times a week meets this objective. Our serial hormonal testing suggests that about 20% of women with POI will ovulate over four months of weekly sampling.

POI? What are the advantages and limitations of this strategy? A: The first question is whether they want to be a parent or not. Some women tell me they never got ’the motherhood gene’ and never wanted to have children. Oocyte donation obviously is not for them. The next question is whether the couple is interested in hightech approaches. Some couples have financial or ethical concerns; oocyte donation is not for them. For such couples, the options include adoption, foster children, or deciding to live child free. For couples who have the means and the desire to adopt high-tech approaches, oocyte donation works extremely well. There is some evidence to suggest a higher incidence of pregnancy complications for this method, and a minimal increase in the rate of birth malformations. However, for most couples these risks are not of great enough magnitude to decide against the approach. Embryo donation is another high-tech approach that can work well and cost less.

Women with spontaneous 46 XX POI have intermittent and unpredictable ovarian function that can continue for decades. There is one report in literature of a woman developing POI at age 27 and having a normal delivery at age 44. The emotional shock of the diagnosis can be quite severe and most women need to process the associated grief, which takes time. We know that the grief of losing a loved one takes about three years to get to the resolution phase. Many women tell me that getting this diagnosis was like losing a loved one, losing the children they hoped to have in the usual manner. It also takes time for the husband and wife to get on the same page with regard to next steps. I suggest that couples should consider permitting some time for natural conception while they process the grief. This needs to be individualized, but in view of the level of grief I have seen in these women, I suggest considering a processing period of three years before moving forward. Obviously, this is not a hard and fast rule, just a place to start considering how to move forward.

Q: With the lack of sufficient evidence on several aspects of POI, would you like to see more studies being done in any specific area? A: Absolutely! This is a rare condition made up of a host of ultra rare diseases. To make substantial research progress, we need to develop an international consortium with a registry that will provide insights into the natural history of this disorder and a base for controlled trials. This work has been supported by the Intramural Research Program, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

Q: Do you recommend ART to all patients, since several studies have demonstrated that oocyte donation is the best strategy to achieve pregnancy in women with

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FEATURED MINI-REVIEW Metformin before and during ART effective in non-obese PCOS women Metformin is used to help induce ovulatory menstrual cycles in oligo-amenorrheic patients with polycystic ovary syndrome (PCOS). A recent multicenter, prospective, randomized, double-blind study reports that 12 weeks of metformin treatment prior to and during IVF/ICSI cycles, as opposed to placebo, substantially increases pregnancy and live birth rates in non-obese PCOS patients.

administered till the hCG injection day, in both normal weight and obese women with PCOS who were scheduled to undergo IVF. The scientists did not find any significant benefit of metformin pretreatment before conventional IVF/ICSI cycles in terms of improvement in stimulation or clinical outcomes. However, pregnancy rates were found to be increased with pretreatment in normal weight PCOS patients. A more recent meta-analysis of randomized trials by Johnson (Australian and New Zealand Journal of Obstetrics and Gynaecology, 2011) suggested metformin and clomiphene to be effective first-line therapeutic strategies for non-obese women with anovulatory PCOS owing to similar effectiveness in terms of CPR and LBR.

Sigrun Beate Kjøtrød from the Department of Gynaecology and Obstetrics, Fertility Clinic, St. Olavs Hospital, Trondheim University Hospital, Norway, and coworkers, conducted the study on 150 PCOS patients with BMI <28 kg/m2 to investigate the influence of metformin on the clinical pregnancy rate (CPR). The subjects were administered with 2,000 mg metformin per day or identical placebo for ≥12 weeks before and during the long protocol IVF/ICSI till the day of testing pregnancy. CPR was considered the primary outcome measure, while spontaneous pregnancy rates during pretreatment period and live birth rates (LBR) were secondary measures.

In order to address the difference in opinion regarding the inclusion of metformin as part of the primary treatment in women with PCOS having anovulatory infertility, the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) sponsored a workshop in 2008. Not supporting the routine use of metformin for ovulation induction, based on the data from recent research, the released consensus statement recommends its use only in women with glucose intolerance.

Prior to the IVF treatment, 15 (20.3%) and eight (10.7%) spontaneous pregnancies were observed in the metformin and placebo groups. Among 112 women who underwent the IVF procedure, there was a difference in CPR (39.3% vs. 30.4%; 95% CI=−8.6 to 26.5) although similar biochemical pregnancy rates (42.9%) were noted. LBR was found to be 37.5% and 28.6% in the metformin and placebo groups, respectively (95% CI=−8.4 to 26.3). Based on the intention-to-treat analyses (n=149), the researchers noted significantly greater overall CPR (50.0% vs. 33.3%; 95% CI=−1.1 to 32.3; P=0.0391) and LBR (48.6% vs. 32.0; 95% CI=1.1 to 32.2; P=0.0383) in the metformin group compared to those on placebo. However, the insulin sensitizer was not found to have an influence on ART outcome, per se.

In an editorial, Nestler (Fertility and Sterility, 2008) differed on the consensus statement of ESHRE and ASRM, opining that metformin has the potential to play an important role in the pharmacological treatment of PCOS and should not be restricted to only women with glucose intolerance. Since many studies have reported improved outcomes with respect to pregnancy rates, the author suggested that women with PCOS who have longer timelines for achieving pregnancy form a ‘welldesigned subset’ in whom metformin, with its gradual onset of action and reduced multiparity risk, could help in re-establishing ovulatory menses and fertility.

Although there were no multiple births or major unexpected safety concerns, gastrointestinal adverse effects occurred more frequently in the metformin than in the placebo group (41% vs. 12%; 95% CI=0.15 to 0.42; P<0.001). These study findings are published in the recent issue of the journal, Human Reproduction.

References 01. Kjøtrød SB, Carlsen SM, Rasmussen PE, et al. Use of metformin before and during assisted reproductive technology in nonobese young infertile women with polycystic ovary syndrome: a prospective, randomized, double-blind, multi-centre study. Hum

Earlier, some of these researchers (Human Reproduction, 2004) analyzed the pretreatment effect of 1,000 mg of metformin twice daily for a minimum of 16 weeks or placebo www.ivfnewsdirect.com

Reprod. 2011 Aug;26(8):2045-53. More references available online

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ART Antisperm antibodies may not adversely influence ART pregnancy rates parameters such as concentration, motility, and strict morphology.

Literature searches on the link between direct semen antisperm antibody (ASA) levels and treatment outcomes in ART cycles have not yielded clear results. Now, a systematic review suggests that semen ASA is not associated with pregnancy rates after IVF or ICSI cycles, thereby indicating the effectiveness of such ART strategies in infertile couples with raised ASA titres.

ASA may hinder the spermâ&#x20AC;&#x2122;s fertilization ability, resulting in immunological infertility, and can also potentially hamper the success of different fertilization techniques. Several studies have been conducted to determine the effectiveness of various treatments, such as corticosteroids, vitamin D3, IUI, IVF, and ICSI, for improving reproductive results in infertile couples with positive ASA, but there is still a lack of consensus on the best approach.

Armand Zini from the Division of Urology, Department of Surgery, McGill University, Montreal, Canada, and colleagues, conducted the systematic review using the Medline database, and short-listed 10 IVF and 6 ICSI studies to investigate the impact of ASA on pregnancy rates after IVF/ICSI. The ASA cut-off levels were heterogeneous in the study. The analysis demonstrated that the combined OR for failing to achieve pregnancy with IVF and ICSI were 1.22 (95% CI=0.84 to 1.77) and 1.00 (95% CI=0.72 to 1.38), respectively, in patients with positive semen ASA. The OR for the combined results of IVF and ICSI cycles was found to be 1.08 (95% CI=0.85 to 1.38). Based on the study findings, the scientists concluded that IVF/ICSI pregnancy rates are not affected by the presence of ASA in semen. The findings of the study are published in the online issue of Human Reproduction.

Although the current study findings indicate that IVF/ ICSI pregnancy rates may not be associated with ASA, the researchers suggest the need for further welldesigned prospective studies to validate the results by using appropriate cut-off values of ASA. Furthermore, there is also a need for continuing research on the sperm and oocyte antigenicity, apart from male and female reproductive tract immunology, to enhance knowledge on the underlying mechanisms for immunologic infertility. This will, in turn, aid in developing new, cost-effective, and specific treatment for such infertile couples. References

Similar results were reported in another recent retrospective study by Zini et al (Journal of Reproductive Immunology, 2011). The researchers analyzed consecutive IVF and IVF/ICSI cycles, in which the patients underwent seminal test for ASA within 6 months before ART cycle. The clinical pregnancy rates in ASA positive couples did not significantly vary from ASA negative samples (42% vs. 52%; OR=1.45; 95% CI=0.63 to 3.30; P>0.05). The findings revealed that reproductive outcomes were not linked to direct ASA levels or sperm

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01. Zini A, Fahmy N, Belzile E, Ciampi A, Al-Hathal N, Kotb A. Antisperm antibodies are not associated with pregnancy rates after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2011 Jun;26(6):1288-95. 02. Zini A, Lefebvre J, Kornitzer G, et al. Anti-sperm antibody levels are not related to fertilization or pregnancy rates after IVF or IVF/ICSI. J Reprod Immunol. 2011 Jan;88(1):80-4. 03. Bubanovic I, Najman S, Kojic S. Immunomodulatory Treatment for Infertile Men with Antisperm Antibodies. Fertil Steril. 2004;81(3):S7-31:20.

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January - March 2012

FSH administration during hCG trigger may improve oocyte developmental competence in IVF cycles The increase in the success rates of ART over the past few decades has been mainly attributed to improved laboratory conditions and ovarian stimulation optimization, with less attention directed towards strategies for enhancing final oocyte maturation. A recent study, touted to be the first, randomized, double blind, placebo-controlled clinical trial to alter the ovulation trigger to enhance the developmental competence of oocytes in IVF cycles, has reported a statistically significant improvement in fertilization after a follicle stimulating hormone (FSH) bolus administration at the time of human chorionic gonadotropin (hCG) trigger, compared to placebo.

pregnancy or ongoing/live birth rates. Earlier, a prospective randomized study by Kovacs et al (Fertility and Sterility, 2008) reported that both recombinant and urinary hCG offers similar serum and follicular hormonal environments for oocyte maturation during the final stages. The researchers also showed that both types of hCG have similar IVF outcome parameters, and thus could be considered as equally effective. Although hCG is generally used for final oocyte maturation in ART cycles, its extended luteotrophic effect has shown to increase the levels of estradiol and progesterone, which may lead to the development of ovarian hyperstimulation syndrome (OHSS). In order to reduce the risk of OHSS, various studies (Sismanoglu et al, Journal of Assisted Reproduction and Genetics, 2009; Itskovitz-Eldor et al, Human Reproduction, 2000) have suggested that GnRH agonist/antagonist may be an alternative to hCG in selected patient population.

Julie D Lamb and coworkers from the University of California, San Francisco, conducted the study to investigate whether additional FSH bolus administration at the time of hCG trigger improves the oocyte developmental competence in IVF cycles. Published in the journal Fertility and Sterility, the study included 188 women (age=25-40 years; mean age=36.2 years) undergoing infertility treatment with long agonist suppression IVF protocol. The subjects were randomly categorized into two groups: group 1 (n=95) was administered FSH bolus at the time of hCG trigger, while group 2 (n=93) received placebo. The primary outcome was fertilization rate, and the secondary outcomes included oocyte recovery, implantation rate, and clinical and ongoing pregnancy/live birth rates. The key findings are listed in Table 1.

An earlier report by the Practice Committee of the American Society for Reproductive Medicine (Fertility and Sterility, 2008) has recommended the following doses of hCG and GnRH agonist for triggering ovulation of a maturated follicle in anovulatory women undergoing IVF: •• Purified hCG dose of 5,000-10,000 IU, IM or SC, or recombinant hCG dose of 250 μg SC •• GnRH agonist, i.e., leuprolide 500 μg SC or triptorelin 200 μg SC. This option should not be used in women who received GnRH during gonadotropin therapy, and in those with hypogonadotropic amenorrhea

Table 1: Primary and secondary outcomes in FSH+hCG and placebo+hCG Outcomes

Group 1 (%)

Group 2 (%)

Fertilization

Likelihood of Oocyte Recovery

Clinical Pregnancy Rate

56.8

46.2

Ongoing/Live Birth Rate

51.6

43.0

Several previous trials, which have aimed to lower ovarian hyperstimulation, have indirectly assessed the benefits of FSH administration through GnRH agonist use for final oocyte maturation, and have found that pituitary surge of FSH and LH occur due to the GnRHa trigger. It has been proposed that FSH may act synergistically, providing a favorable environment for final oocyte maturation and ovulation, directly by promoting plasminogen activity or indirectly by improving responsiveness to hCG via follicular environment modulation. It has also been found that FSH plays an important role in keeping gap junctions

The study findings showed statistically significant improvement in rates of fertilization and the likelihood of oocyte recovery in group 1 compared to those receiving placebo, whereas no substantial variation was observed between the two groups with respect to clinical www.ivfnewsdirect.com

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open between the oocyte and cumulus cells, and hence may play a vital role in signaling pathways.

January - March 2012

blind, placebo-controlled trial. Fertil Steril. 2011 Apr;95(5):1655-60. 02. Kovacs P, Kovats T, Bernard A, Zadori J, Szmatona G, Kaali SG. Comparison of serum and follicular fluid hormone levels with recombinant and urinary human chorionic gonadotropin during in

With the increasing need for identifying newer strategies to enhance ART success rates, the current study findings could serve as a method for improving oocyte competence using physiology-based modifications. Although the results suggest FSH supplementation during hCG trigger as a promising option to improve IVF outcomes, further research is warranted to validate the findings and also understand the underlying mechanism of the potential benefits of FSH administration.

vitro fertilization. Fertil Steril. 2008 Dec;90(6):2133-7. 03. Sismanoglu A, Tekin HI, Erden HF, Ciray NH, Ulug U, Bahceci M. Ovulation triggering with GnRH agonist vs. hCG in the same egg donor population undergoing donor oocyte cycles with GnRH antagonist: a prospective randomized cross-over trial. J Assist Reprod Genet. 2009 May;26(5):251-6. 04. Itskovitz-Eldor J, Kol S, Mannaerts B. Use of a single bolus of GnRH agonist triptorelin to trigger ovulation after GnRH antagonist ganirelix treatment in women undergoing ovarian stimulation for assisted reproduction, with special reference to the prevention

References

of ovarian hyperstimulation syndrome: preliminary report: short

01. Lamb JD, Shen S, McCulloch C, Jalalian L, Cedars MI, Rosen MP.

communication. Hum Reprod. 2000 Sep;15(9):1965-8.

Follicle-stimulating hormone administered at the time of human

05. Practice Committee of American Society for Reproductive

chorionic gonadotropin trigger improves oocyte developmental

Medicine. Use of exogenous gonadotropins in anovulatory women:

competence in in vitro fertilization cycles: a randomized, double-

a technical bulletin. Fertil Steril. 2008 Nov;90(5 Suppl):S7-12.

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January - March 2012

Japanese study reports lower ectopic pregnancy risk with frozen-thawed single blastocyst transfer The Joint Society of Obstetricians and Gynaecologists of Canada-Canadian Fertility and Andrology Society Clinical Practice Guidelines Committee (JOINT SOGC-CFAS), in 2010, recommended the adoption of single blastocyststage embryo transfer when good quality blastocysts are available, considering the association with better implantation and live birth chances compared to cleavagestage embryos. Now, a retrospective study reports that single frozen-thawed blastocyst transfers are associated with significantly lower risk of ectopic pregnancy (EP) when compared to fresh cycles.

linked to increased EP rate. The reduced risk noted during fresh cycles could be due to the levels of supraphysiologic progesterone, which lower uterine contractility and increase implantation in the uterine cavity. Although several recent studies have not found any enhanced risk with frozen transfer, the investigators have cautioned clinicians on considering the possibility of the increased risk before deciding on the treatment. An earlier retrospective study by Jun and Milki (Fertility and Sterility, 2007) demonstrated that there is no substantial rise in the EP rate following frozen blastocyst transfer when compared to fresh cycles. In the program, blastocysts were cryopreserved and transferred using abdominal ultrasound guidance, 1-1.5 cm short of the fundus. The scientists observed an EP rate of 2.8% and 1.8% in the frozen and fresh blastocyst groups, respectively.

Osamu Ishihara, Professor, Department of Obstetrics and Gynecology, Faculty of Medicine, Saitama Medical University, Japan, and coworkers, retrospectively analyzed the Japanese registry of assisted reproductive medicine to compare EP rates between fresh and frozen-thawed single blastocyst transfer cycles. The results showed that the incidence of EP was 0.81% (84/10,312 clinical pregnancies), 1.8% (25/1,361 clinical pregnancies), and 1.4% (19/1,352 clinical pregnancies) in frozen-thawed, fresh IVF, and fresh ICSI single blastocyst transfer cycles, respectively. Following age group stratification, a variation in the EP rate, ranging from 0.56% to 0.97%, was noted after frozen-thawed single blastocyst transfers.

Evidently, studies report contradictory results on the chances of EP after frozen blastocyst transfer, but there is a lot of heterogeneity among the trials. Hence, it is imperative to conduct well-designed studies, while considering the potential biases, to validate the association between fresh/frozen blastocyst transfer and EP. Additionally, research is warranted to investigate how factors such as different hormonal milieu, embryo implantation potential, and technical issues of IVF contribute to the increased risk of EP after ART procedures.

Based on the study findings, published in the recent online issue of Fertility and Sterility, the researchers concluded that there is a significant reduction in the rates of EP with frozen-thawed compared to fresh blastocyst stage single embryo transfer cycles.

References

A 2008 Practice Committee report of the American Society for Reproductive Medicine (Fertility and Sterility) identified ART as one of the major risk factors for the occurrence of EP. Similarly, in a 2008 presentation by Pinborg at European Society of Human Reproduction and Embryology Campus course, LĂźbeck, blastocyst transfer and frozen embryo transfer were indicated as probable risk factors of EP.

01. Ishihara O, Kuwahara A, Saitoh H. Frozen-thawed blastocyst transfer reduces ectopic pregnancy risk: an analysis of single embryo transfer cycles in Japan. Fertil Steril. 2011 May;95(6):1966-9. 02. Jun SH, Milki AA. Ectopic pregnancy rates with frozen compared with fresh blastocyst transfer. Fertil Steril. 2007 Sep;88(3):629-31. 03. Practice Committee of American Society for Reproductive Medicine. Medical treatment of ectopic pregnancy. Fertil Steril. 2008 Nov;90(5 Suppl):S206-12. 04. Keegan DA, Morelli SS, Noyes N, Flisser ED, Berkeley AS, Grifo JA.

Assessing the risk factors for EP after ART, Hye Jin and Chang Suk (Current Opinion in Obstetrics and Gynecology, 2010) found that frozen embryo transfer, assisted hatching, large embryo transfer volume, zygote intrafallopian tube transfer, and deep fundal transfer are

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Low ectopic pregnancy rates after in vitro fertilization: do practice habits matter? Fertil Steril. 2007 Sep;88(3):734-6. 05. Chang HJ, Suh CS. Ectopic pregnancy after assisted reproductive technology: what are the risk factors? Curr Opin Obstet Gynecol. 2010 Jun;22(3):202-7.

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INFERTILITY Better pregnancy rates reported with single post-ovulation IUI compared to single pre-ovulation IUI A recent prospective cohort study has reported that cycle pregnancy rates (CPR) are better with single intrauterine insemination (IUI) timed post-ovulation, compared to pre-ovulation, in patients with non-male infertility. However, pre-ovulation double IUI, compared to single IUI, was seen to offer better CPR for male factor-related infertility.

24.13; P=0.06) and 1.2 (95% CI=0.43 to 3.33; P=0.779) for etiologies of male and non-male factor infertility, respectively. There is a lack of consensus on the frequency and accuracy of IUI timing following hCG trigger, in ART cycles. An earlier retrospective cohort study by Kucuk (Journal of Assisted Reproduction and Genetics, 2008) suggests that the observation of follicular rupture before IUI may provide pregnancy rates comparable to natural fertility. The study involved 121 couples with male factor subfertiliy and 296 couples with unexplained infertility. During the study, 578 cycles were analyzed and follicle rupture evaluated using transvaginal ultrasonography after IUI. The clinical pregnancy rate in follicle rupture evident and non-evident groups was 23.5% and 8.8%, respectively. The study findings concluded that IUI should be performed when follicular rupture is detected.

Mohamad E Ghanem from the Mansoura Integrated Fertility Center, Egypt, and co-researchers, conducted the study to investigate the association of CPR with the following: •• Timing of single IUI at 36±2 hours post-human chorionic gonadotropin (hCG) administration, pre- or post-ovulation •• Number of IUI (single or double) in pre-ovulatory patients with male, anovulatory, and unexplained infertility The study involved 1,146 first-stimulated cycles in infertile couples who were stimulated with human menopausal gonadotropin (hMG), clomiphene citrate (CC) or sequential CC-hMG, and monitored using transvaginal ultrasound. Ovulation was triggered by 10,000 IU hCG when the primary follicle reached ≥18 mm mean diameter, and IUI timed at 36±2 hours. Single IUI alone was performed in post-ovulatory women, whereas women in their pre-ovulatory stage were sequentially randomized to single or double IUI, with CPR being the end point.

A more recent prospective study by Tonguc et al (Fertility and Sterility, 2010) has reported no difference with respect to rates of pregnancy with single IUI (24 hour) and double IUI (12 and 36 hours); although the preferred IUI timing was 36 hours post-hCG administration. However, the researchers recommended that 24-hour IUI may be ideal for demanding situations. Studies have reported numerous methods to decide the optimal timing for IUI such as luteinizing hormone (LH) testing in urine and blood, ultrasound scanning for ovulation detection, basal body temperatures (BBT) chart, hCG administration, combination of hCG administration and LH detection, and gonadotropinreleasing hormone (GnRH) agonist administration. A recent review by Cantineau and colleagues (Cochrane Database Systemic Reviews, 2010) reported no significant difference in the treatment outcome among the different timing methods. It was suggested that the choice of treatment should depend on hospital facilities, convenience of patients, medical staff, costs, and dropout levels.

The study findings, published in the journal Human Reproduction, were as follows: •• The overall CPR among the whole cohort was 10.1%. •• CPR was 11.7% with evidence of ovulation, prior to IUI, and 6.7% without ovulation (OR=1.85; 95% CI=1.12 to 3.06; P=0.015). •• OR was 1.26 (95% CI=0.52 to 2.95; P=0.82) for male factor infertility and 2.24 (95% CI=1.23 to 4.08; P=0.007) for non-male factor infertility. •• On comparing the CPR between single and double IUI in pre-ovulatory patients, OR was 1.9 (95% CI=0.76 to 4.7; P=0.22) for all cycles, and 4.667 (95% CI=0.9 to www.ivfnewsdirect.com

Several studies have reported variable CPR, ranging 18

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factor infertility subjects, and double IUI in patients with male factor infertility if ovulation did not occur during the first IUI. Furthermore, the researchers suggest the need for larger prospective randomized trials comparing single and double IUI by evaluating controlled timing in relation to ovulation in male factor infertility patients.

from 4.5% to 22%, in stimulated cycles for different etiologies of infertility. The plausible reason for higher CPR in infertile couples with non-male etiologies could be the presence of fertile semen when IUI is timed after follicular rupture. However, the availability of oocyte with inadequate survival rates remains the limiting factor in such patients. The reasons for the conďŹ&#x201A;icting outcomes based on the CPRs in single and double IUI, as supposed by the scientists of the current study, are that the techniques have not been compared with respect to the same infertility etiologies, and are not associated with ovulation during IUI.

References 01. Ghanem ME, Bakre NI, Emam MA, et al. The effects of timing of intrauterine insemination in relation to ovulation and the number of inseminations on cycle pregnancy rate in common infertility etiologies. Hum Reprod. 2011 Mar;26(3):576-83. 02. Kucuk T. Intrauterine insemination: is the timing correct? J Assist Reprod Genet. 2008 Aug;25(8):427-30.

The current study is touted to be the first to investigate the effect of single and double IUI timing with regard to ovulation in various infertility etiologies in a single cohort. It has been reported that maximum outcome with minimal cost and effort could be attained by selective single IUI, timed post-ovulation in non-male

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January - March 2012

03. Tonguc E, Var T, Onalan G, et al. Comparison of the effectiveness of single versus double intrauterine insemination with three different timing regimens. Fertil Steril. 2010 Sep;94(4):1267-70. 04. Cantineau AE, Janssen MJ, Cohlen BJ. Synchronised approach for intrauterine insemination in subfertile couples. Cochrane Database Syst Rev. 2010 Apr 14;(4):CD006942.

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CRYOPRESERVATION Study reports effective blastocyst cryopreservation using closed blastocyst vitrification system Reproduction, 2011) reported that day 5 and day 6 blastocysts, obtained from biopsied embryos, can be successfully cryopreserved using closed CBS-VIT HS straws. The researchers analyzed 100 warming cycles in patients who had undergone aneuploidy screening and/or preimplantation genetic diagnosis (PGD) in fresh cycles. A total of 131 blastocysts were vitrified using closed CBS-VIT HS straws, among which the morphological survival and transfer rates were 83.2% and 79.4%, respectively. The corresponding clinical pregnancy rates after SET and DET were found to be 19.2% (15/78) and 38.5% (5/13). The study findings showed better survival in day 5 blastocysts (90.4%) when compared to day 6 blastocysts (70.8%; P<0.01); however, no significant difference in the rates of survival were noted between early cavitating (89.2%) and full/expanded blastocysts (93.3%).

A recent study has reported the effectiveness of a closed blastocyst vitrification (CBS-VIT) high security (HS) device for the cryopreservation of blastocysts from early cavitating to expanded blastocyst stage. Lisbet Van Landuyt and coworkers from the Center for Reproductive Medicine, Universitair Ziekenhuis Brussel, Belgium, investigated the outcome of the closed blastocyst vitrification system with respect to blastocyst score prior to cryopreservation in single embryo transfers (SET). Vitrification of supernumerary blastocysts of IVF/ ICSI patients with fresh day-5 transfer was performed using CBS-VIT HS straws. The rates of morphological survival and transfer with respect to the blastocyst score and the day of vitrification were evaluated in 759 warming cycles, while the pregnancy rates were assessed in 530 SET and 156 double embryo transfer (DET) cycles. In SET cycles, the rates of implantation per embryo transfer (ET) were investigated based on the quality of blastocyst and day of cryopreservation.

Earlier, Kuwayama et al (Reproductive BioMedicine Online, 2005) indicated the effectiveness of the closed over open vitrification system in eliminating the risk of embryo contamination while freezing and storing without compromising on the rates of survival and development, in vitro and in vivo.

The study findings, published in the journal Human Reproduction, are as follows: •• Immediate morphological survival and transfer rate per warmed blastocyst were 77.8% (921/1,185) and 70.7% (838/1,185), respectively. •• Rates of survival were higher in day 5 with early blastocysts (86.7%) compared to full (78.7%) or expanded blastocysts (72.7%). •• Survival rate for day 6 blastocysts was reduced (70.1%) compared to day 5 blastocysts (80.6%; P<0.001). •• Clinical pregnancy rates after SET and DET were found to be 16.4% and 24.4%, respectively, and corresponding ongoing pregnancy rates 14.2% and 20.5%. •• Ongoing multiple pregnancy rate after DET was 21.8% (7/32). •• Implantation rates were substantially lower in day 5 early blastocysts (10.6%) than in advanced blastocysts (17.5%; P<0.05), while the implantation potentials were similar for day 5 and day 6 blastocysts (14.3% vs. 13.7%).

Previous studies have not reported transmission of infectious diseases through cryopreservation and storage of embryos; however, such risks may arise under experimental conditions. The aseptic method of vitrification using open devices may necessitate sterilization of liquid nitrogen and a secondary enclosure for storage. Thus, recent studies have suggested the use of hermetically sealed containers, as it has the advantage of both vitrification and storage of embryos under ‘closed’ conditions. The requirement for stringent sanitary conditions for preserving oocytes or embryos free of infectious agents has led to the development of the closed vitrification system, without direct exposure of embryos or semen to liquid nitrogen. The closed system may require a carrier to transport the maximum heat transfer rate to the specimen, as the sample has no direct contact with liquid nitrogen. Closed containers have also shown to attain

Similarly, another study by some researchers (Human www.ivfnewsdirect.com

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this limited allowance of heat transfer by design (being ultrathin and containing microvolumes) and selection of appropriate material.

January - March 2012

vitrification outcomes. References 01. Van Landuyt L, Stoop D, Verheyen G, et al. Outcome of closed

The cryopreservation of blastocysts is challenging owing to its multicellular structure, size, and presence of blastocele. Since single blastocyst transfer is being practiced more often in ART, there is an increasing need for a simple, convenient, and accurate approach for cryostorage of surplus blastocyts. With the current study demonstrating the superiority of the closed over the open system of vitrification, the former has the potential to enhance ART success rates. However, further studies are necessary to validate the safety of the procedure in view of its novelty. Furthermore, training embryologists in this technique would also play a major role in improving the

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blastocyst vitrification in relation to blastocyst quality: evaluation of 759 warming cycles in a single-embryo transfer policy. Hum Reprod. 2011 Mar;26(3):527-34. 02. Van Landuyt L, Verpoest W, Verheyen G, et al. Closed blastocyst vitrification of biopsied embryos: evaluation of 100 consecutive warming cycles. Hum Reprod. 2011 Feb;26(2):316-22. 03. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online. 2005 Nov;11(5):608-14. 04. Kader AA, Choi A, Orief Y, Agarwal A. Factors affecting the outcome of human blastocyst vitrification. Reprod Biol Endocrinol. 2009 Sep 16;7:99.

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GENETICS Study on vertical transmission of hepatitis B infection to offspring through germ cells The magnitude of the risk of vertical transmission of hepatitis B virus (HBV) infection through oocyte or embryo is not clearly known. A new study demonstrates the potential association between the presence of HBV DNA in oocytes and embryos, and the serum HBV DNA levels in the women’s serum and the mothers’ infection status, respectively, thereby indicating the transmission of the infection via germ cells to the offspring.

embryos were noted in the group with high serum HBV DNA concentrations compared to those with low levels (P=0.004 vs. P=0.002). •• Higher oocyte positivity rates were noted in women whose mothers were infected with HBV than those with uninfected mothers. •• HBsAg expression was found in 8.7% oocytes and 14.1% embryos (P=0.34).

Xiao-Ling Hu and coworkers from the Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, China, investigated the possibility of HBV transmission via germ cells from parents to offspring. The study was performed on 250 oocytes taken from hepatitis B surface antigen (HBsAg) seropositive women and 578 embryos from couples in whom at least one of the parent was HBsAg seropositive. Fluorescence in situ hybridization technique was used to detect HBV DNA in the nuclei of oocytes and embryos, while immunofluorescence and real-time PCR were used to analyze HBsAg expression and serum HBV DNA levels, respectively. The researchers examined the duration of HBV infection in women and their mothers’ serum HBsAg status.

Based on the above results, the researchers concluded that the embryos infected with HBV were maternally or paternally dependent. Similar findings were demonstrated in another recent study by Nie et al (Fertility and Sterility, 2011). The study results that indicate the probability of vertical HBV transmission through germ line are represented in Table 1. The researchers did not find HBV DNA, RNA, and HBsAg in 135 oocytes and embryos retrieved from 39 seronegative couples. Contrary to this, Lou et al (The Journal of International Medical Research, 2010) reported that vertical transmission via oocytes is not the main route of HBV infection. Immunohistochemical investigation of eggs of carrier pregnant women, showed HBsAg-positive ova in only one woman (out of 68 subjects); however, the newborn tested negative for serum HBV markers after 3 days of birth. Only one neonate (out of 68 neonates) tested positive for serum HBV markers 3 days after birth, while the ova was HBsAg negative.

The study findings, published in Human Reproduction, are listed below. •• HBV DNA was present in 9.6% of oocytes and 14.4% of embryos. •• Similar rates of HBV DNA-positive embryos were observed when the man (21.3%), woman (13.1%), or both (14.9%) tested HBsAg seropositive. •• Significantly greater rates of positivity in oocytes and

As per a 2008 report by the Practice Committee of the

Table 1: HBV infection in chronic HBV carriers

Parameters evaluated and tested positive

Male HBsAg-positive/ female HBsAg-negative couples

Embryos of male HBsAgpositive/female HBsAgnegative couples

Male HBsAg-negative/ female HBsAg-positive couples

Oocytes and embryos of male HBsAg-negative/female HBsAg-positive couples

HBV DNA

3 of 18

13 of 84

3 of 14

15 of 71

HBV RNA

9 of 13

39 of 52

8 of 17

30 of 63

HBsAg

6 of 10

13 of 20

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American Society for Reproductive Medicine (Fertility and Sterility, 2008), the development of chronic HBV infection occurs in 2-6% adults, 30-60% children, and 90% infants exposed to HBV. An earlier review by RangerRogez et al (Pathologie Biologie, 2002) indicated that the rate of transmission to the offspring is estimated to be 90% in serum HBV DNA-positive mothers and 10-30% in serum HBV DNA-negative mothers.

References 01. Hu XL, Zhou XP, Qian YL, Wu GY, Ye YH, Zhu YM. The presence and expression of the hepatitis B virus in human oocytes and embryos. Hum Reprod. 2011 Jul;26(7):1860-7. 02. Nie R, Jin L, Zhang H, Xu B, Chen W, Zhu G. Presence of hepatitis B virus in oocytes and embryos: a risk of hepatitis B virus transmission during in vitro fertilization. Fertil Steril. 2011 Apr;95(5):1667-71. 03. Lou H, Ding W, Dong M, et al. The presence of hepatitis B surface antigen in the ova of pregnant women and its relationship with

The ASRM Committee report also states that although the risk of viral hepatitis transmission is possible in ART, its exact magnitude is not clearly known. Furthermore, high-risk infertile couples interested in undergoing fertility treatment should be tested for HBsAg in order to decrease the probable risk of transmission to the uninfected partner, fetus, and disease-free gametes and embryos.

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January - March 2012

intra-uterine infection by hepatitis B virus. J Int Med Res. 2010 JanFeb;38(1):214-9. 04. Practice Committee of American Society for Reproductive Medicine. Hepatitis and reproduction. Fertil Steril. 2008 Nov;90(5 Suppl):S226-35. 05. Ranger-Rogez S, Alain S, Denis F. Hepatitis viruses: mother to child transmission [in French]. Pathol Biol. 2002 Nov;50(9):568-75.

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NEWS ESHRE defines poor response to ovarian stimulation The lack of a clear consensus on the definition of poor ovarian response (POR) in ART has made it difficult to compare results of various studies conducted to improve ovarian response; in turn hindering the identification of poor responders and selection of an optimal ovarian stimulation strategy. In a first realistic endeavor, the European Society of Human Reproduction and Embryology (ESHRE) working group proposed a standardized, simple, and reproducible definition for POR. The ESHRE consensus is published in the recent issue of the journal, Human Reproduction.

The Group indicated that such a comprehensive criteria will help researchers select a homogenized population for future studies, and lower bias due to spurious POR definitions, thus facilitating the comparison of results for obtaining accurate conclusions. Several studies have attempted to arrive at an objective definition of POR. In one such study, Kailasam et al (Human Reproduction, 2004) proposed the inclusion of the degree of ovarian stimulation in the definition of POR during IVF cycles in women <40 years of age. According to the study, patients should be considered poor responders if they require >3,000 IU FSH cumulative dose to obtain sufficient follicles. Cancellation at ≥300 IU FSH/day is linked to a substantially worse prognosis and was also considered as a criterion to define POR. The scientists indicated that their findings may help in the development of a standardized definition for poor response.

On behalf of the ESHRE working group, Anna Pia Ferraretti from the S.I.S.Me.R Reproductive Medicine Unit, Bologna, Italy, and coworkers, arrived at a consensus on the definition of POR, and recommend that at least two of the following must be met to consider patients as poor responders: •• Advanced maternal age or presence of other risk factors of POR •• History of POR •• Abnormal ovarian reserve test (ORT)

With the absence of uniform criteria for defining POR, the recent consensus could assist both clinicians and researchers improve ovarian response and treatment outcomes, as well as develop and evaluate protocols for its prevention and management.

Apart from the above three criteria, the ESHRE group suggested the following: •• Patients can be considered as poor responders following two POR episodes after maximal stimulation, if advanced maternal age or abnormal ORT is not a factor. •• Although one stimulated cycle is necessary for diagnosing POR, patients with advanced age or abnormal ORT may be categorized as poor responders, as both the factors suggest the possibility of decreased ovarian reserve, and therefore serve as markers of stimulation cycle outcomes. Such patients should be classified as ‘expected poor responders.’

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References 01. Ferraretti AP, La Marca A, Fauser BC, et al; ESHRE working group on Poor Ovarian Response Definition. ESHRE consensus on the definition of ’poor response’ to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011 Jul;26(7):16161624. 02. Kailasam C, Keay SD, Wilson P, Ford WC, Jenkins JM. Defining poor ovarian response during IVF cycles, in women aged <40 years, and its relationship with treatment outcome. Hum Reprod. 2004 Jul;19(7):1544-7.

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January - March 2012

Semen washing does not eliminate risk of HPV infection Conventional sperm recovery procedures are hypothesized to depend on the migration of motile sperms away from washed epithelial cells and immotile sperms, thus leading to the elimination of infected epithelial cells and enabling virus-free sperm preparations. Although this seems biologically plausible, a recent cross-sectional clinical study highlights that conventional sperm selection techniques may not entirely remove the risk of HPV infection.

reported in an earlier study by Olatunbosun et al (Obstetrics & Gynecology, 2001). Semen samples collected from 85 subjects were detected for viral DNA sequence using nested PCR. HPV was detected in 21 of 32 participants with identifiable lesions and six out of 53 without the lesions (P<0.001). Only two cases showed reduction in cellular HPV DNA level below noticeable levels with swim-up washings of all 27 pre-wash sperm cells with HPV. Considering the results, the researchers suggested that with the prevalence of HPV infection even in apparently healthy subjects, the semen of prospective donors should be tested for HPV DNA, and those with positive tests should not be included in the donation program.

Carlo Foresta and colleagues from the Clinical Pathology Section & Centre for Male Gamete Cryopreservation, University of Padova, Italy, evaluated the efficacy of three sperm washing protocols: swim-up, discontinuous Ficoll density gradient, and simple wash (centrifugation), for eliminating HPV-infected cells from semen samples of infertile patients. The study involved 32 infertile patients confirmed to be semen HPV positive through polymerase chain reaction (PCR) and in situ hybridization techniques on sperm and exfoliated cells. Semen analysis and in situ hybridization were carried out prior and subsequent to the three protocols in the cross-sectional study to detect HPV; the results were statistically analyzed using two tailed Student’s t-test.

With HPV being frequently observed in urethral and seminal swabs of normal men, in 2008, the Practice Committee of the American Society for Reproductive Medicine (ASRM) reported the lack of a consistent approach for screening donors and decreasing the risk of the infection. Due to the presence of HPV in semen as cellfree virus and in epithelial cells, sperm-wash protocols have the potential to decrease the infectiousness of HPV-infected men. Furthermore, ASRM recommends that female partners undergoing sperm donation cycles should undergo screening for sexually transmitted infections. This could help in taking necessary precautions for reducing the risk of transmission to offspring and uninfected partners, apart from identifying preexisting infections and documenting new transmissions during ART procedures.

Outcome measures included the assessment of sperm parameters and HPV presence in samples before and after sperm selection procedures. HPV DNA on sperm and exfoliated cells was found in fifteen samples. Infected sperm with a mean percentage of positivity (24.7% ± 8.9%) was noted to be greater than exfoliated cells (13.8% ± 4.3%) in all the native samples. No difference was observed in the percentage of infected cells and the number of infected samples with sperm washing centrifugation. However, slight decrease in the number of infected samples was induced by the Ficoll and swimup protocols (30 and 26, respectively). Based on the study findings, the researchers suggested the need for careful monitoring of HPV infected patients as there is an increased risk of infection transmission and its associated adverse effects on fetal development.

References 01. Foresta C, Pizzol D, Bertoldo A, Menegazzo M, Barzon L, Garolla A. Semen washing procedures do not eliminate human papilloma virus sperm infection in infertile patients. Fertil Steril. 2011 Apr 30. [Epub ahead of print] 02. Olatunbosun O, Deneer H, Pierson R. Human papillomavirus DNA detection in sperm using polymerase chain reaction. Obstet Gynecol. 2001 Mar;97(3):357-60. 03. Practice Committee of American Society for Reproductive Medicine. Guidelines for reducing the risk of viral transmission

Similar findings with respect to the ineffectiveness of sperm washing methods in eliminating HPV sperm infection were

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during

fertility

treatment.

Fertil

Steril.

2008

Nov;90(5

Suppl):S156-62.

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January - March 2012

British IVF data shows association between number of oocytes retrieved and live birth rate Touted as the first study to report the association between live birth rates (LBR) and number of eggs retrieved, a UKbased study has indicated that around 15 oocytes should be retrieved in an IVF cycle to maximize LBR. The study proposes the number of retrieved oocytes as a robust indicator of the IVF success rates.

between 15 and 20, and reducing when the number was more than 20. However, in an earlier study that evaluated the link between number of oocytes retrieved and pregnancy rates during ART, Yoldemir and Fraser (Archives of Gynecology and Obstetrics, 2010) concluded that the number of retrieved oocytes do not affect pregnancy outcomes, with respect to clinical and ongoing pregnancy rates. Women were classified based on oocyte count into 3 groups: those with <5 eggs, 5-10 oocytes, and 11-15 eggs. No variation was seen in pregnancy rates among the three groups.

Arri Coomarasamy, a clinical reader and consultant in reproductive medicine and surgery at the University of Birmingham, UK, and coworkers, analyzed data of 400,135 IVF cycles from April 1991 to June 2008, obtained from the Human Fertilisation and Embryology Authority (HFEA). Using a prediction model that replicates the current IVF practice, the scientists determined the chances of live birth for a given oocyte number, based on age stratification. Further, a nomogram was developed with the help of the model for predicting LBR after IVF, on the basis of women’s age and oocyte count.

Although ovarian reserve tests, such as antral follicle count (AFC) and anti-Mullerian hormone (AMH) level measurement, may be good predictors of ovarian response and oocyte yield after stimulation, LBR cannot be accurately predicted. The authors of the current study suggest that their nomogram could be used by clinicians to estimate LBR by using egg yield predicted by AFC and AMH. The findings thus have the potential to complete the prognostic chain in estimating the success rates of IVF treatment. Furthermore, the researchers also propose that the aim of the standard stimulation should be to retrieve 10 to 15 oocytes for obtaining the best treatment outcomes, since there is an increased risk of ovarian hyperstimulation syndrome if the ooctye count exceeds 20.

The key study findings, published in the recent issue of the journal Human Reproduction, are listed below. •• Median oocyte number retrieved per cycle was 9 (inter-quartile range [IQR]=6-13) •• Overall LBR was 21.3% per fresh IVF cycle •• LBR predicted in women of different age groups, in whom 15 oocytes were retrieved between 2006 and 2007, is given in Table 1 Table 1: Live birth rate in women with 15 oocytes Women Age (years)

Predicted LBR (%)

18–34

35–37

J, Coomarasamy A. Association between the number of eggs and

38–39

live birth in IVF treatment: an analysis of 400 135 treatment cycles.

≥40

References 01. Sunkara SK, Rittenberg V, Raine-Fenning N, Bhattacharya S, Zamora

Hum Reprod. 2011 Jul;26(7):1768-74. 02. First study to investigate how many eggs are needed to achieve a live birth after IVF finds 15 is the perfect number [press release].

The scientists noted a strong non-linear relationship between the number of retrieved oocytes and LBR; with LBR increasing with the oocyte number, peaking with around 15 eggs, plateauing when the eggs retrieved were

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UK: ESHRE; May 11, 2011. Accessed May 20, 2011. 03. Yoldemir T, Fraser IS. The effect of retrieved oocyte count on pregnancy outcomes in an assisted reproduction program. Arch Gynecol Obstet. 2010 Mar;281(3):551-6.