To the Editor:

We read the article by Meuleman et al. (1) with interest. The high incidence of endometriosis (47%) and other pelvic pathology (29%) in this cohort makes a strong case for offering laparoscopy and hysteroscopy to couples with “unexplained infertility” (a diagnosis of exclusion). This diagnosis however requires confirmation of tubal integrity, and the issue is whether laparoscopy should be the first-line investigation to establish tubal patency. The authors did not say how many women in their cohort had a completely normal pelvis following laparoscopy and hysteroscopy.

Their data, put in another way, showed that 53% of the women who under went laparoscopy did not have endometriosis. It is in an effort to minimize the risks and costs associated with laparoscopy that NICE (2) recommends that hysterosalpingogram be considered as the first-line test for tubal patency, and laparoscopy be considered only for women with “co-morbidity.” Although the definition of co-morbidity is fairly non-specific, it generally refers to women whose clinical history may suggest the presence of potential pelvic pathology. This includes those who have had previous pelvic infection, previous ectopic pregnancy, known endometriosis, etc. By limiting laparoscopy to this cohort, fewer women are exposed to the risks associated with this procedure and its potential cost implications.

Also, as interesting as it is to know that laparoscopy revealed a high incidence of endometriosis and other “fertility-reducing pathologies” among the cohort of women studied, the authors failed to discuss the outcome of the treatment of these pathologies. We must never lose sight of the fact that the ultimate aim of infertile women undergoing any type of treatment is to take home a baby. Until this is achieved, the patients may be thankful for having their asymptomatic endometriosis resected, but will remain dissatisfied and consider the treatment a failure should they not have a baby at the end. It will be interesting to know how many women had a live birth following surgery in this cohort.

Krithiga Ilangavan, MRCOG
Kingston Hospital NHS Trust
Kingston Upon Thames, United Kingdom

Emmanuel Kalu, MRCOG
Assisted Concepton Unit
Queen Mary’s Hospital
London, United Kingdom

References
1. Meulemann C, Vandenabeele B, Fieuws S et al. High Prevalence of endometriosis in infertile women with normal ovulation and normospermic partners. Fertil Steril 2009, 92;1:68-74.

2. National Collaboration Centre for Women’s and Children’s Health. Clinical Guideline 11:fertility: assessment and treatment for people with fertility problems. UK, NICE, 2004.

Endometriosis: Is laparoscopy justified without previous ultrasonogram and MRI?

To the Editor:

In the article by Meuleman et al. (1), I appreciated the new understandings brought by the authors and the unexpected demonstration that pelvic pain was not explained by the existence of endometriotic lesions in 46% of cases in infertile women.

Nevertheless, I am surprised about the last sentence of their abstract, “Reproductive surgery is indicated in infertile women belonging to the study population….” Nothing in the article supports this assertion, neither the statistical results nor the references. However, the following criticisms are more important:

1) Whereas so many details are credited to the expertise of the surgical team, nothing is mentioned about the ultrasonographer’s competence. Still d’Hooghe and Timmerman themselves had written that the results of ultrasonograms are dependent on the experience and interest of the ultrasonographer (2). The progress made in discovering nodular lesions of endometriosis and the abnormal position of pelvic organs due to adhesions, when the ultrasonographer is talented, cannot be ignored.

2) Magnetic resonance imaging (MRI) is never mentioned. Even if the team does not have access to the necessary equipment, the information contributed by this technique is so well recognized (3) that it should have been mentioned, since the indications for surgery with its possible side effects are not the same if the lesions discovered necessitate it or if they might just be improved by surgery.

3) More surprisingly, the existence or absence in the patients of adenomyosis associated with endometriosis was never mentioned in work from a team that has written a remarkable article on adenomyosis and infertility (2). It is possible that the diagnosis of adenomyosis being difficult for the ultrasonographer, and as no MRI had been used, the authors decided not to raise the question, but this deficiency seriously affects the conclusions of the work, as the internal adenomyotic lesions are not accessible to laparoscopic surgery! Nevertheless, as hysteroscopy was a mandatory part of the investigations, it is difficult to understand that a possible role of adenomyotic lesions has never been at least hypothesized.

Bearing in mind all these uncertainties, it appears that the conclusions in the article are at least too abrupt, as authors insist on the advantages of surgery, which is today subject of discussion because its dangers have been hidden for such a long time and because the progress of MRI and ultrasonography are now well established, in parallel with the good results of assisted reproductive technology. In the August issue of Fertility and Sterility, Chapron et al. have written, “When the clinical examination (questioning and pelvic exam) suggests deep infiltrating endometriosis we feel that the first additional investigation should be transvaginal ultrasonography. In this context, the discovery of an ovarian endometrioma at transvaginal ultrasonography should prompt the practitioner to search for severe lesions (intestinal and/or ureteral involvement) rather than programming surgery immediately.” (4).

It will be a pleasure if the authors’ reply shows that my criticisms were unjustifiably excessive.

Jean Belaisch, M.D.
Department of Gynaecology and Obstetrics
Saint Vincent de Paul Hospital
Paris, France

References
1. Meuleman C, Vandenabeele B, Fieuws S, Spiessens C, Timmerman D, D’Hooghe T. High prevalence of endometriosis in infertile women with normal ovulation and normospermic partners. Fertil Steril. 2009 Jul;92(1):68-74.

2. Devlieger R, D’Hooghe T, Timmerman D. Uterine adenomyosis in the infertility clinic. Hum Reprod Update. 2003 Mar-Apr;9(2):139-47.

3. Kunz G, Beil D, Huppert P, Noe M, Kissler S, Leyendecker G. Adenomyosis in endometriosis–prevalence and impact on fertility. Evidence from magnetic resonance imaging. Hum Reprod. 2005 Aug;20(8):2309-16.

4. Chapron C , Pietin-Vialle C , Borghese B, Davy C, Foulot H, Chopin N. Associated ovarian endometrioma is a marker for greater severity of deeply infiltrating endometriosis. Fertil Steril 2009 Aug ; 92(2): 453-57.

The Authors Respond:

We thank colleagues Belaisch, Ilangavan and Kalu for their interest in our work (1).

Our conclusion that reproductive surgery is indicated in infertile women with regular menstrual cycles whose male partners have normal sperm quality is justified. Firstly, significant pelvic (including endometriosis), tubal or uterine pathology was observed in 2/3 of our patients and can be interpreted as significant comorbidity and justification for surgery according to the NICE guidelines (2). Secondly, a completely normal hysteroscopy and laparoscopy were only observed in 1/3 of our patients (1), but in this “normal” group diagnostic hysteroscopy and laparoscopy can still be clinically justified to confirm the diagnosis of unexplained infertility, since a normal hysterosalpingography does not exclude significant pelvic pathology (3).

Although a lot of progress has been made in the ultrasound diagnosis of endometriosis with respect to ovarian endometriotic cysts and nodular lesions in the rectovaginal area, and both experience and interest of the ultrasonographer are important in the diagnosis of adenomyosis (4), ultrasound is not reliable in the diagnosis or exclusion of pelvic peritoneal endometriosis found in women with minimal to mild endometriosis (5). Furthermore, some endometriotic nodules are situated higher in the rectosigmoidal area, which is not routinely accessible by transvaginal ultrasound. Finally, the retrospective nature of our study and the variable interval between preoperative ultrasound and surgery may have diluted the diagnostic value of ultrasound in this study. This aspect will be taken care of in our ongoing prospective cohort study.

We disagree with Dr Belaisch that hysteroscopy is useful for the diagnosis of endometriosis (no published data supporting this statement) or that magnetic resonance imaging (MRI) is needed for the diagnosis of adenomyosis, as several studies have shown that ultrasound diagnosis of adenomyosis is as reliable as MRI diagnosis (4-6). Adenomyosis was not a relevant issue in our study for the following reasons: Firstly, the relationship between adenomyosis and infertility is not properly quantified; secondly, adenomyosis cannot be treated by surgery or by medication in women who want to become pregnant. Therefore, in our study, adenomyosis was not a factor of importance in deciding for or against surgery.

Clearly, MRI is not a first-line investigation in this study population of infertile women whose husbands have normal sperm (5). MRI or other imaging techniques like intravenous pyelogram or bowel contrast radiography are indicated and used in our center when more extensive endometriosis with impact on bowel, bladder and/or ureter is suspected based on clinical examination and ultrasound imaging (5, 7). Furthermore, based on randomized controlled trials, excisional endometriosis surgery has been proven to improve fertility in women with minimal to mild disease and to reduce pain in women with mild to moderate endometriosis (5), justifying surgery in this population. It is important to emphasize that about 50% of subfertile women with endometriosis also had pain, and that cotreatment of both subfertility and pain is important in this group. The use of assisted reproductive technology (8) cannot be considered as a first-line infertility treatment of endometriosis (5). The possible risks of laparoscopic endometriosis surgery need to be acknowledged, but can be considered to be minimal when this surgery is done by experienced reproductive gynecological surgeons, in the context of a multidisciplinary center of excellence for endometriosis (9). Indeed, the integration of endometriosis care in such centers may be the only way to prevent overtreatment of women with endometriosis by assisted reproduction, potentially driven by the industrialization of assisted reproduction worldwide, and a useful way to embed endometriosis surgery in a larger context of reproductive medicine (9). Overall, the goal of infertility treatment in women with endometriosis should be to first surgically remove endometriosis and surgically treat associated pelvic pathology, and then to decide on expectant management, controlled ovarian stimulation with or without intrauterine insemination or assisted reproduction (8), based on female age, tubal condition after surgery, and duration of infertility. This strategy is currently evaluated in a prospective outcome study.

Christel Meuleman, M.D.^a
Carla Tomassetti, M.D.^a
Dirk Timmerman, M.D., Ph.D.^b
Thomas D’Hooghe, M.D., Ph.D.^a

Leuven, Belgium

References
1. Meuleman C, Vandenabeele B, Fieuws S, Spiessens C, Timmerman D, D’Hooghe T
High prevalence of endometriosis in infertile women with normal ovulation and normospermic partners. Fertil Steril. 2009 Jul;92(1):68-74

2. National Collaboration Centre for Women’s and Childrens’ Health. Clinical Guideline 11: fertility: assessment and treatment for people with fertility problems. UK, NICE, 2004. http://www.nice.org.uk/nicemedia/pdf/CG011fullguideline.pdf

3. Bosteels J, Van Herendael B, Weyers S, D’Hooghe T. The position of diagnostic laparoscopy in current fertility practice Hum Reprod Update 2007; 13:477-85. Epub 2007 Jun 11.

4. Devlieger R, D’Hooghe T, Timmerman D. Uterine adenomyosis in the infertility clinic. Hum Reprod Update. 2003 Mar-Apr;9(2):139-47.

5. Kennedy S, Bergqvist A,Chapron C, D’Hooghe T, Dunselman G, Greb R, Hummelshoj L, Prentice A, Saridogan E. On behalf of the ESHRE Special Interest Group for Endometriosis and Endometrium Guideline Development Group.(the manuscript was prepared by the first author; all other authors contributed equally and are listed in alphabetical order) ESHRE Guideline for the Diagnosis and Treatment of Endometriosis. Hum Reprod 2005;20:2698-704.

6. Bazot M, Cortez A, Darai E, Rouger J, Chopier J, Antoine JM, Uzan S. Ultrasonography compared with magnetic resonance imaging for the diagnosis of adenomyosis: correlation with histopathology. Hum Reprod. 2001 Nov;16(11):2427-33.

7. Chapron Ch , Pietin-Vialle C , Borghese B, Davy C, Foulot H, Chopin N. Associated ovarian endometrioma is a marker for greater severity of deeply infiltrating endometriosis. Fertil Steril 2009 Aug ; 92 (2) : 453-7.

8. Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, Sullivan E, and van der Poel S, on behalf of ICMART and WHO.The International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) Revised Glossary on ART Terminology, 2009.Hum Reprod 2009;24:2683-7.

9. D’Hooghe TM, Hummelshoj L. Multi-disciplinary centres/networks of excellence for endometriosis management and research: a proposal. Hum Reprod 2006;21(11):2743-8.

To the Editor:

We read with great interest the manuscript by Fanfani et al., which reports their experience with laparoscopic treatment of bowel-infiltrating endometriosis (1). The authors analyzed their patients in regards to operative long-term complications, pain relief and recurrence rate, and suggest a surgical management algorithm. However, women with bowel endometriosis, besides having gastrointestinal symptoms and pain, frequently suffer from infertility.

We mostly agree with the proposed treatment algorithm, but believe some data regarding fertility after surgery should be pointed out and considered.

Fanfani et al. reported a 27.3% pregnancy rate in women subjected to discoid resection suffering from infertility (1). These data appear below the 44% previously reported by Mohr et al. in 9 patients subjected to discoid resection (2). By contrast, no information regarding the pregnancy rate is given for the control group undergoing segmental bowel resection (2).

It should be noted, that segmental resection by laparotomy yields fertility rates between 39.4% and 50% (3,4). Laparoscopic colorectal resection for endometriosis has been shown to be feasible (5,6), and recent findings have convincingly shown that, in this setting of patients, laparoscopy yields better results when compared to traditional laparotomy (7). Ferrero et al. reported fertility rates as high as 43% in 21 infertile women after bowel resection, and Darai et al. reported a 45.5% pregnancy rate among 22 women wishing to conceive after laparoscopic segmental colorectal resection (7,8) (Table 1).

Click on table to enlarge.

The mechanism by which endometriosis impairs fertility has not yet been clearly defined and probably ges beyond a simple mechanical explanation. Infertility could be explained by functional alterations of the peritoneal environment affecting gamete, embryo or fallopian tube function, in addition to the important pelvic anatomical abnormalities (9). Remorgida et al. reported that endometriotic tissue remains in over 40% of women subjected to full-thickness disc resection. These authors hypothesized that endometriotic tissue can spread laterally from the primary nodule following the enteric nervous system, thus explaining the low rate of radical discoid resection (10). The persistence of microscopic endometriotic foci could have an effect on fertility.

Until the pathophysiological relationship between endometriosis and infertility is fully understood, surgeons will be required to balance their “conservative attitude” with surgical radicality. Currently, we believe that it would be of great interest to have the fertility results achieved in the segmental resection group in this important study, in order to better tailor the proposed treatment algorithm and apply it to women with infertility.

Marco Calcagno, M.D.
Filippo Bellati, M.D.
Maria Pastore, M.D.
Francesco Plotti, M.D.
Innocenza Palaia, M.D.
Pierluigi Benedetti Panici, M.D.
Department of Obstetrics and Gynecology
University “La Sapienza”
Rome, Italy

References
1. Fanfani F, Fagotti A, Gagliardi ML, Ruffo G, Ceccaroni M, Scambia G, Minelli L. Discoid or segmental rectosigmoid resection for deep infiltrating endometriosis: a case-control study. Fertil Steril 2009; doi:10.1016/j.fertnstert.2009.03.066

2. Mohr C, Nezhat FR, Nezhat CH, Seidman DS, Nezhat CR. Fertility consideration in laparoscopic treatment of infiltrative bowel endometriosis. J Soc Laparosc Surg 2005;9:16–24.

3. Coronado C, Franklin RR, Lotze EC, Bailey HR, Valdes CT. Surgical treatment of symptomatic colorectal endometriosis. Fertil Steril 1990;53:411–6.

4. Bailey HR, Ott MT, Hartendorp P. Aggressive surgical management for advanced colorectal endometriosis. Dis Colon Rectum 1994;37:747–53.

5. Possover M, Diebolder H, Plaul K, Schneider A. Laparoscopically assisted vaginal resection of rectovaginal endometriosis. Obstet Gynecol 2000;96:304 –7.

6. Redwine DB, Wright JT. Laparoscopic treatment of complete obliteration of the cul-de-sac associated with endometriosis: long-term follow- up of en bloc resection. Fertil Steril 2001;76:358–65.

7. Ferrero S, Anserini P, Abbamonte LH, Ragni N, Camerini G, Remorgida V. Fertility after bowel resection for endometriosis. Fertil Steril. 2009;92(1):41-6.

8. Darai E, Marpeau O, Thomassin I, Dubernard G, Barranger E, Bazot M. Fertility after laparoscopic colorectal resection for endometriosis: preliminary results. Fertil Steril 2005;84:945–50.

9. Witz CA, Burns WN. Endometriosis and infertility: is there a cause and effect relationship? Gynecol Obstet Invest 2002;53 Suppl 1:2–11.

10. Remorgida V, Ragni N, Ferrero S, Anserini P, Torelli P, Fulcheri E. How complete is full thickness disc resection of bowel endometriotic lesions? A prospective surgical and histological study. Hum Reprod 2005;20:2317–20.

The Authors Respond:

We appreciate the letter by Calcagno et al. regarding indications for surgery for deep infiltrating endometriosis (DIE) and posed questions about our study (1).

We proposed a management algorithm for DIE (1) underlining the opportunity to treat only patients with symptoms or asymptomatic patients with severe bowel stenosis. Both discoid and segmental rectosigmoid resection were found to be safe and feasible, with superimposable clinical outcomes (1). In this study, we reported a 27.3% spontaneous pregnancy rate in women referred preoperatively for infertility who underwent discoid resection.

Calcagno et al. suggest that this result appears lower than those reported by other authors on discoid (2) and/or segmental rectosigmoid resections (3). However, in these studies (2,3) the fertility data are not homogeneous and the number of cases is very low. In particular, the reported pregnancy rate is not separated into spontaneous and assisted pregnancy, and there are no data regarding preoperative infertility. It is impossible to assume that in all study populations infertility is due only to the presence of endometriosis, thus suggesting that only spontaneous pregnancy after radical endometriosis ablation can be considered a result of endometriosis-related infertility. Nevertheless, we have previously reported our series of laparoscopic colorectal segmental resection for DIE, showing a pregnancy rate comparable with other studies (41.6%), but with the vast majority (80%) assisted by a reproductive technique (4). In the segmental resection group (controls) of the present study (1), the pregnancy rate is almost the same, with a global pregnancy rate of 42%, of which 75% used an assisted reproductive technique. These data confirm that our results in terms of post-operative pregnancy rate are similar to those reported in the literature.

In addition, Calcagno et al. suggest that desire for fertility should be taken into account in the surgical treatment algorithm of DIE patients, and that the persistence of microscopic endometriosis foci after discoid resection showed by Remorgida et al. (5) could have a negative effect on fertility. Stepniewska et al. (6) showed that the presence of bowel infiltration by endometriosis negatively influences the reproductive outcome in women with endometriosis-associated infertility, concluding that the complete removal of endometriosis seems to offer better results in terms of post-operative fertility with respect to patients not undergoing bowel endometriosis ablation.

In our opinion, currently there are not sufficient data to affirm that discoid full-thickness resection of rectosigmoid endometriosis results in a lower pregnancy rate compared with segmental resection. Similar post-operative results in terms of pregnancy rate seem to offer to the surgeon an opportunity to freely choose between the two techniques, when indicated. To this end, we performed a case-control study between discoid and segmental resection confirming the role of discoid resection in small nodules (< 3 cm).

We hope that future prospective studies will be focused on endometriosis-related infertility and major surgery in order to provide answers to this enigmatic problem.

Francesco Fanfani, M.D.¹
Anna Fagotti, M.D.¹
Maria Lucia Gagliardi, M.D.¹
Luca Minelli, M.D.²
Giovanni Scambia, M.D.¹

²Department of Obstetrics and Gynecology
“Sacro Cuore” Hospital
Negrar, Italy

References
1. Fanfani F, Fagotti A, Gagliardi ML, Ruffo G, Ceccaroni M, Scambia G, Minelli L. Discoid or segmental rectosigmoid resection for deep infiltrating endometriosis: a case-control study. Fertil Steril 2009;doi:10.1016/j.fertnstert.2009.03.066.

2. Mohr C, Nezhat FR, Nezhat CH, Seidman DS, Nezhat CR. Fertility consideration in laparoscopic treatment of infiltrative bowel endometriosis. J Sic Laparosc Surg 2005;9:16-24.

3. Ferrero S, Anaserini P, Abbamonte LH, Ragni N, Camerini G, Remorgida V. Fertility after bowel resection for endometriosis. Fertil Steril 2009;92:41-6.

4. Minelli L, Fanfani F, Fagotti A, Ruffo G, Ceccaroni M, Mereu L, Landi S, Pomini P, Scambia G. Laparoscopic colorectal resection for bowel endometriosis: fesibility, complications, and clinical outcome. Arch Surg 2009;144:234-9.

5. Remorgida V, Ragni N, Ferrero S, Anserini P, Torelli P, Fulcheri E. How complete is full thickness disc resection of bowel endometriosis lesions? A prospective surgical and histological study. Hum Reprod 2005;20:2317-20.

6. Stepniewska A, Pomini P, Bruni F, Mereu L, Ruffo G, Ceccaroni M, Scioscia M, Guerriero M, Minelli L. Laparoscopic treatment of bowel endometriosis in infertile women. Hum Reprod 2009;24:1619-25.

To the Editor:

I should like to comment on the interesting report of Morales et al. (1) describing prenatally detected cases of androgenetic/biparental mosaicism.

Two mechanisms of origin have been discussed for case 1 that represent a mixture of androgenetic 46,XX and normal biparental 46,XY cells. One alternative involves dispermic fertilization of an oocyte and formation of a triploid zygote followed by a ‘division without replication’ and missegregation of the paternal pronuclei. In the resulting 2n/n mosaic, endoreduplication of the haploid paternal genome would finally create the diploid androgenetic cell line.

This example resembles our observation of an oocyte with three pronuclei that underwent a premature irregular cytokinesis including one of the pronuclei in the smaller half of the divided cytoplasm (2). We surmised that isolation of the supernumerary pronucleus by premature cytokinesis might be relevant for diploidization of triploid zygotes and also for the development of haploid/diploid mosaics or diploid uniparental cell lines. Though the oocyte described by us obviously had two maternal pronuclei, the corresponding mechanism is also applicable to a dispermic oocyte. Here, one half of the divided ooplasm could receive the maternal pronucleus and one of the paternal pronuclei, giving rise to a diploid biparental cell line. The second paternal pronucleus within the other half of the ooplasm could undergo endoreduplication and create an androgenetic cell line. This concept differs from the model suggested by Morales et al. (1) in one decisive point, i.e., a triploid one-cell zygote will not be formed because there is a cytoplasmic division before (not without) replication.

The alternative mechanism of origin for case 1 is based on the assumption that a normally fertilized oocyte fuses with another fertilized but ‘empty’ oocyte. In other words, the second cell is supposed to contain no maternal chromosomes. To the best of my knowledge, there has been no case without chromosomes among the thousands of female gametes that became available for analysis in the course of assisted reproduction (3) and I agree with Golubovsky (4) who already stated that convincing evidence for the existence of anuclear oocytes does not exist.

Morales et al. (1) further mention fertilization of a normal oocyte by a diploid spermatozoon as a third but unlikely possibility. In a previous review I have compiled supportive evidence for this event as a mechanism of origin for diandric triploids (5). A possible restriction, however, is the fact that formation of a diploid paternal pronucleus may mainly occur after intracytoplasmic sperm injection because diploid spermatozoa are particularly found in oligozoospermic men.

Therefore, I wonder whether the cases presented by Morales et al. (1) have resulted from natural conceptions or from assisted reproduction and also, whether a diploid spermatozoon could have played a role in their second case. This would eliminate the necessity of postulating a failure of DNA replication in the female pronucleus followed by normal replication in the embryo.

I appreciate the efforts of Morales et al. (1) who tried to clarify the origin of the observed abnormalities and hope that information obtained from assisted reproduction will serve as a useful contribution.

Bernd E. Rosenbusch, Ph.D.
Department of Gynaecology and Obstetrics
University of Ulm
Ulm, Germany

References
1. Morales C, Soler A, Badenas C, Rodríguez-Revenga L, Nadal A, Martínez JM, et al. Reproductive consequences of genome-wide paternal uniparental disomy mosaicism: description of two cases with different mechanisms of origin and pregnancy outcomes. Fertil Steril 2009;92:393.e5-e9.

2. Rosenbusch BE, Schneider M. Separation of a pronucleus by premature cytokinesis: a mechanism for immediate diploidization of tripronuclear oocytes? Fertil Steril 2009;92:394.e5-e8.

3. Rosenbusch B. The incidence of aneuploidy in human oocytes assessed by conventional cytogenetic analysis. Hereditas 2004;141:97-105.

4. Golubovsky MD. Postzygotic diploidization of triploids as a source of unusual cases of mosaicism, chimerism and twinning. Hum Reprod 2003;18:236-42.

5. Rosenbusch B. Mechanisms giving rise to triploid zygotes during assisted reproduction. Fertil Steril 2008;90:49-55.

The Authors Respond:

We would like to thank Dr. Rosenbusch for his interest in our article and his comments.

The aim of our paper was to describe two prenatal cases of androgenetic/biparental mosaicism diagnosed in our hospital and review in the literature the most suitable mechanisms of origin in each case. We want to clarify that the different hypotheses proposed have been previously reported by different authors. Moreover, both cases we present have resulted from natural conception, since the couples were never referred for fertility problems.

We agree with Dr. Rosenbusch that in the model of dispermic fertilization of an oocyte in case one, a premature irregular cytokinesis could take place in the oocyte with three pronuclei (one maternal and two paternal), giving rise to a diploid biparental cell and an androgenetic cell. However, premature cytokinesis is considered a very rare abnormality and has only occasionally been seen in unfertilized oocytes (1).

With respect to the existence of “empty” oocytes, it has theoretically been related with the formation of complete moles (2) and it was suggested as a possibility in the model of Surti et al. (3).

Fertilization by a diploid spermatozoon could be a possible scenario. However, this type of event is more frequently related to oligo-crypto-azoospermic males, while normozoospermic individuals produce diandric triploids mainly by dispermy (4).

In summary, multiple hypotheses could be postulated to explain the origin of our cases. We believe that the models we propose are the most probable ones, taking into account that in both cases conception was achieved naturally in couples without fertility problems. As Dr. Rosenbusch states, the description of such cases is essential to clarify the origin of this type of genomic abnormalities.

Carme Morales, Ph.D.
Anna Soler, Ph.D.
Irene Mademont-Soler, Ph.D.
Aurora Sánchez, M.D., Ph.D.
Servei de Bioquímica i Genètica Molecular
Hospital Clínic
Barcelona, Spain

References
1. Rosenbusch BE, Schneider M. Separation of a pronucleus by premature cytokinesis: a mechanism for immediate diploidization of tripronuclear oocytes? Fertil Steril 2009;92:394.e5-8.

2. Fan JB, Surti U, Taillon-Miller P, Hsie L, Kennedy GC, Hoffner L et al. Paternal origins of complete hydatidiform moles proven by whole genome single-nucleotide polymorphism haplotyping, Genomics 2002;79:58-62.

3. Surti U, Hill LM, Dunn J, Prosen T and Hoffner L. Twin pregnancy with a chimeric androgenetic and biparental placenta in one twin displaying placental mesenchymal dysplasia phenotype. Prenat Diagn 2005; 25:1048-56.

4. Egozcue S, Blanco J, Vidal F, Egozcue J. Diploid sperm and the origin of triploidy. Hum Reprod 2002;17:5-7.

To the Editor:

We read with interest the article by Francavilla et al. published in the recent issue of the Journal (1). The authors set out to “analyze the effectiveness of intrauterine insemination with or without mild ovarian stimulation in male subfertility using a prospective cross-over design.”

They justify cross-over design (CD) with the expectation to prevent bias caused by variability in fertility potential of participating couples. Variation in patient characteristics can be considered a problem with conventional “parallel group” randomized trials, requiring substantial number of patients in each group in order to estimate reliably the magnitude of any treatment effect.

Intuitively, CD allowing within-patient comparisons may seem as a better option. However, the role of the CD in infertility research has been controversial (2-4). When pregnancy or live birth is the outcome of interest, participants who conceive in the former period are censored and become unavailable for the alternative treatment in the latter period, preventing within patient comparison. Such participants decrease the effective sample size and cast shadow on the advantage of CD (5). On the other hand, it has been argued that CD may provide an unbiased estimate of the treatment effect if data are analyzed appropriately (2, 4).

Regardless of which side of the debate one stands, repeated observations on a subject tend to be more alike than observations on different subjects. The standard error is smaller when such “multiplicity” is ignored and leads to biased estimate of treatment effect.

Given the figures reported by Francavilla et al., the majority of participants must have undergone several treatment cycles and contributed to trial data several times. Moreover, participants were allocated to three different treatments, namely, intrauterine insemination (IUI) alone, IUI with ovarian stimulation and timed intercourse in a natural cycle (NI) for a maximum of six treatment cycles (1). The trial therefore could be considered as a 3 x 6 cross-over trial, further complicating analysis.

We wonder if the inclusion of the NI arm is justified, given the trial objectives, how multiplicity was addressed at the analysis stage, and whether the use of Mantel Haenszel chi square test can be justified given the observations were not independent.

Baris Ata, M.D.
William Buckett, M.D.
McGill Reproductive Centre
McGill University Health Centre
Montreal, Quebec, Canada

References
1. Francavilla F, Sciarretta F, Sorgentone S, Necozione S, Santucci R, Barbonetti A, et al. Intrauterine insemination with or without mild ovarian stimulation in couples with male subfertility due to oligo/astheno- and/or teratozoospermia or antisperm antibodies: a prospective cross-over trial. Fertil Steril 2009;92:1009-11.

2. Makubate M. The Place of Crossover Designs in Infertility Trials. In: Statistics. Vol. Doctor of Philosophy in Medical Statistics. Glasgow: University of Glasgow, 2009.

3. Khan KS, Daya S, Collins JA, Walter SD. Empirical evidence of bias in infertility research: overestimation of treatment effect in crossover trials using pregnancy as the outcome measure. Fertil Steril 1996;65:939-45.

4. McDonnell J, Goverde AJ, Vermeiden JP. The place of the crossover design in infertility trials: a maximum likelihood approach. Hum Reprod 2004;19:2537-44.

5. Daya S. Methodological issues in infertility research. Best Pract Res Clin Obstet Gynaecol 2006;20:779-97.

The Authors Respond:

I thank Dr. Ata and Dr. Buckett for their interest in our paper.

I agree that some concerns address the use of cross-over studies in the field of subfertility due to possible bias in estimation of treatment effect. However, simulation models have shown that crossover and parallel designs will produce essentially the same statistical estimates of treatment effect and percentage of pregnancies (1).

Reply to specific queries:

1. The inclusion of natural intercourse (NI) arm was necessary because the primary objective of the study was to determine the effectiveness of IUI with or without mild ovarian stimulation, i.e., versus NI.

2. Multiplicity was addressed using survival analysis with discrete timing of events (“proportional odds”) to evaluate the effect of treatments over time (2,3). In contrast to Cox proportional hazards, the cycle number entered as a nominal variable to handle time and treatments as a time-dependent explanatory variable (to take into account that each woman might have had several cycles of
treatments and that LBR drop as a result of selection).

As stated in the text, each couple was offered up to six treatment cycles, three with IUI+COH and three with IUI besides three natural cycles with NI. It means 3 x 3 and not 3 x 6 cross-over trial, as interpreted by Dr. Ata and Dr Buckett.

3. The Mantel Haenszel chi square was used to compare the treatment outcome, including IUI and IUI+COH, in different categories of male subfertility. Therefore, this analysis was justified, because observations were independent.

Felice Francavilla, M.D.
Department of Internal Medicine
Andrologic Unit
University of L’Aquila
L’Aquila, Italy

References
1. Cohlen BJ, te Velde ER, Looman CW, Eijckemans R, Habbema JD. Crossover or parallel design in infertility trials? The discussion continues. Fertil Steril 1998;70:40-5.

2. Kalbfleisch J, Prentice RL. The Statistical Analysis of Failure Time Data. Wiley, New York 1980.

3. Cohlen BJ, te Velde ER, van Kooij RJ, Looman CW, Habbema JD. Controlled ovarian hyperstimulation and intrauterine insemination for treating male subfertility: a controlled study. Hum Reprod 1998;13:1553-8.

To the Editor:

We read with great interest the paper published recently by Elizur et al. in the September 2009 edition of Fertility and Sterility (1). The authors compared retrospective data of two different protocols used to perform in vitro maturation treatment (IVM) based on physician preference: only micronized 17β-estradiol (6-12 mg/day) for endometrial preparation or human menopausal gonadotropins (HMG) 150 IU/day to induce both endometrial preparation and mild follicle stimulation. Both protocols started between day 6-10 of the cycle and the main outcome measure was the endometrial thickness before oocyte retrieval. We would like to congratulate the authors: we believe that endometrial receptivity is concerning in IVM treatment and studies trying to improve implantation rates should also have the endometrium as target.

However, the authors did not include in the discussion section one study that also evaluated two different endometrial preparations proposed for IVM in PCOS women: starting estradiol 14 days after the menstruation (the planned day of oocyte retrieval: 6 mg/day or 10 mg if endometrial thickness less than 5 mm) or a sequentially increasing dosage of estradiol beginning on the first day of menstrual flow (2 mg/day for 4 days, 4 mg/day for 4 days and then 6 mg/day) (2). Using estradiol since the beginning of the menstrual cycle all women achieved an endometrial thickness greater than 8 mm and the endometrial thickness on the day 14 was 9.96 ± 2.22mm; which is considerably higher than the observed in the two groups on the study of Elizur et al. (1): 7.7 ± 1.8mm and 7.3 ± 1.7 (HMG and estradiol groups). Since the main outcome measure was the endometrial thickness, this previous report should be considered.

A recent publication by Zhao et al. (3) also demonstrated better results in IVM cycles when using estradiol since the beginning of the cycle (days 3-5): the endometrial thickness on the day of embryo transfer was 8.72 ± 0.85 mm, with an implantation rate of 15.42% (3) against an implantation rate of 9.6% when estradiol was used only after the day of oocyte retrieval described by other center (4), both retrieving oocytes from unstimulated ovaries of PCOS women. The implantation rate described by Elizur et al. (1) in the HMG group (15.0%) was very close to that described using estradiol since the beginning of the cycle by Zhao (3), which suggests that both strategies similarly improve endometrial receptivity.

Wellington P. Martins Ph.D.
Carolina O. Nastri, M.D.
Rosana M. Reis, Ph.D.
Rui A. Ferriani, Ph.D.
Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina de Ribeirão Preto
Universidade de São Paulo
Sao Paulo, Brazil

References
1. Elizur SE, Son WY, Yap R, Gidoni Y, Levin D, Demirtas E et al. Comparison of low-dose human menopausal gonadotropin and micronized 17beta-estradiol supplementation in in vitro maturation cycles with thin endometrial lining. Fertil Steril 2009;92:907-12.

2. Martins Wde P, dos Reis RM, Ferriani RA, de Araujo CH, Nastri CO, Filho FM. Endometrial preparation for in vitro oocyte maturation: early use of estrogen increases endometrial tissue and requires lower daily dosage: a cross over trial in ‘mock’ cycles. J Assist Reprod Genet 2006;23:241-6.

3. Zhao JZ, Zhou W, Zhang W, Ge HS, Huang XF, Lin JJ. In vitro maturation and fertilization of oocytes from unstimulated ovaries in infertile women with polycystic ovary syndrome. Fertil Steril 2009;91:2568-71.

4. Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL. In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril 2001;76:936-42.

The Authors Respond:

I wish to thank Martins et al. for their interest in our manuscript and their important letter to the editor. In their study (1) they compared two protocols of estradiol supplementation in polycystic ovary syndrome (PCOS) women undergoing in vitro maturation (IVM) cycles. Patients who received estradiol beginning on the first day of the menstrual flow achieved an endometrial thickness > 8 mm. However, other important cycle characteristics, such as the number of retrieved oocytes, maturation rate, the number of in vivo matured oocytes and pregnancy rates are not mentioned in that study. We have shown (2) that low-dose human menopausal gonadotropin (hMG) supplementation protocol is equivalent to estradiol protocol in terms of endometrial response but is superior to it in terms of the number of in vivo matured oocytes and the maturation rate 24 hours after oocyte retrieval.

These two variables were previously shown (3) to significantly improve embryo quality and pregnancy rates in IVM cycles. Therefore, we believe that when comparing two protocols designated to improve endometrial lining in IVM cycles, one must evaluate the impact on all variables contributing to success in IVM cycles and not only endometrial receptivity. By doing so, we will be able to improve pregnancy and delivery rates in IVM treatments.

Shai E Elizur, M.D.
IVF Unit
Chaim Sheba Medical Center
Tel Hashomer, Israel

References
1. Martins Wde P, dos Reis RM, Ferriani RA, de Araujo CH, Nastri CO, Filho FM. Endometrial preparation for in vitro oocyte maturation: early use of estrogen increases endometrial tissue and requires lower daily dosage: a cross over trial in ‘mock’ cycles. J Assist Reprod Genet 2006;23:241-6.

2. Elizur SE, Son WY, Yap R, Gidoni Y, Levin D, Demirtas E et al. Comparison of low-dose human menopausal gonadotropin and micronized 17beta-estradiol supplementation in in vitro maturation cycles with thin endometrial lining. Fertil Steril 2009;92:907-12.

3. Son WY, Chung J, Demirtas E, Holzer H, Buckett WM, Chian RC, et al. Comparison of cycles programmed for IVM with and without in vivo matured oocytes retrieved. Reprod Biomed Online 2008;17(1):59-67

To the Editor:

We disagree with the results of the trial by Blake et al. (1) with regard to the pharmacokinetics of Crinone and Endometrin.

Subjects in their study were healthy fertile women with normal ovulatory cycles and progesterone levels. Progesterone levels vary considerably in normal menstrual cycles, and dosing occurred day 5-8 (1-day dosing) and 7 days later in the luteal phase (multi-day dosing). Therefore, a significant part of the measured progesterone was most likely endogenously produced.

Even if we assume that none of the measured progesterone was endogenous, the data suggest that the blood sample collection may not have adequately estimated the peak and total systemic exposure for Crinone. The estimates for Crinone total systemic exposure by Blake (1) are substantially lower than previously reported (2) (Crinone label). In the Blake paper (1), the mean area under the curve (AUC_0-24) (Table 2) was 81 ng-h/mL and 264 ng-h/mL following single- and multiple-dose administration, respectively. The corresponding values reported previously were 133 ng-h/mL2 and 167 ng-h/mL (Crinone label, calculated from C_{avg 0-24} data) following single dose and 392 ng-h/mL (Crinone label) following multiple doses. An explanation for this discrepancy is the lack of a 6-hour blood sample. With Crinone, the T_max for progesterone in the previous studies approximated 5-7 hours (compared with 12-13 hours in the present study). The shape of the concentration-time curves for Crinone in figures 1 and 3 supports the conclusion that systemic exposure may not have been adequately estimated.

Also, variability of total exposure was not reported. The coefficients of variation for the AUC_0-24 values for the single dose are calculated to be 52.1%, 50.4% and 51.4% for the insert b.i.d., insert t.i.d. and Crinone, respectively, and 38.8%, 24.3% and 42.7% following multiple doses. Therefore, variances in the pharmacokinetic parameters are similar and substantial for all formulations, resulting in the low power of this study to distinguish between treatments, given the small sample size and non-crossover design.

Furthermore, there are few data to support the assertion that a progesterone level of 10 ng/mL is associated with good outcomes in an IVF cycle. On the contrary, the consensus is that blood progesterone levels do not correlate with pregnancy outcome in an IVF cycle. (3,4)

Finally, the large clinical trial referenced by Blake et al. (5) found a primary efficacy result for ongoing pregnancy of 42% for Crinone, 42% for Endometrin t.i.d., and 39% for Endometrin b.i.d., leading us to question the conclusions of the present study.

Larry S. Abrams, Ph.D.
Brian M. Berger, M.D.
Boston IVF/Harvard Medical School
Quincy, Massachusetts

References
1. Emily J. Blake, Paul M. Norris, Sally Faith Dorfman, James Longstreth, Vladimir I. Yankov. Single and multidose pharmacokinetic study of a vaginal micronized progesterone insert (Endometrin) compared with vaginal gel in healthy reproductive-aged female subject. Fertility and Sterility [epub ahead of print]
DOI: 10.1016/j.fertnstert.2009.06.014

2. Levine H, Watson N. Comparison of the pharmacokinetics of Crinone 8% administered vaginally versus prometrium administered orally in postmenopausal women. Fertility and Sterility, 2000; 73(3): 516-521

3. Chantilis SJ, Zeitoun KM, Patel SI, et al. Use of Crinone vaginal progesterone gel for luteal support in in vitro fertilization cycles. Fertil Steril. 1999;72:823–829.

4. Williams SC, Donahue J, Muasher SJ. Vaginal progesterone therapy during programmed cycles for frozen embryo transfer: an analysis of serum progesterone levels and pregnancy rates. Fertil Steril. 2000;74(suppl 1):S209. Abstract P-363.

5. Doody KJ, Schnell VL, Foulk RA, Miller CE, Kolb BA, Blake EJ, Yankov VI. Endometrin® for luteal phase support in a randomized, controlled, open-label, prospective IVF clinical trial using a combination of Menopur® and Bravelle®. Fertil Steril. 2009; Apr;91(4):1012-7.

The Authors Respond:

We wish to correct the misperceptions stated in the response letter to the pharmacokinetics study of Crinone and Endometrin (1). The results showed that Endometrin rapidly produced higher progesterone serum concentrations, achieved greater systemic exposure, reached steady-state more rapidly, and cleared sooner after termination of therapy than Crinone.

Regarding the comment on time of peak concentration, sustained-release dosage forms are generally intended to yield a concentration-time profile with a broad, flat peak or plateau. Therefore, T_max may have any of a wide range of values, since the maximum observed concentration will be the one with the largest positive random excursion from that plateau’s nominal value. In support of the profile for Crinone being such a plateau, three subjects had T_max at 12 hours or greater and three had T_max at four hours or earlier during the multiple dosing. As displayed in Figure 1 (1), mean Crinone concentrations were uniform over the entire 24-hour dosing interval, thus, focusing on a specific value for the Crinone T_max has little clinical utility. The clinical or pharmacokinetic significance of a difference in mean (or median) Tmax of 6 versus 12 hours is questionable in this setting.

Second, the correspondents question the suitability of the sampling protocol, claiming that the Crinone label gives an AUC_0-24 on Day 1 of 167 ng-hr/mL and after multiple doses, 392 ng-hr/mL, as compared with the values reported in our paper of 81 and 264 ng-hr/mL. However, the value for AUC_0-24 under multiple dosing conditions in the 10-year-old Crinone label is actually 216 ng-hr/mL instead of the 392 cited by the correspondents. The 392 value comes from the table entry for the AUC_0-t, which is not the same as AUC_0-24. AUC_0-24 must be calculated from the C_avg value (i.e., 8.99 ng/mL*24 hours). The AUC_0-t, which is the area to the last quantified or quantifiable concentration, would be expected to be substantially larger than the AUC_0-24. Since 216 is not numerically greater than 264, the argument that not including a six-hour sampling time might explain our “low” observed concentrations is invalid.

Regarding the argument that the high variability (CV) observed in the study precludes drawing any conclusions about differences between treatments; while exposure values were greater with Endometrin, not surprisingly statistical significance was not reached, given the intentionally small subject population. Pharmacokinetics studies designed to elucidate dose-concentration relationships under single or multiple dose conditions typically use 6-8 subjects. This sample size is a compromise between being large enough that the mean value is representative of a larger population, while minimizing unnecessary exposure of healthy people to a medicinal agent. The study design was consistent with standard practice for meeting the study objectives. As stated in the article, “Limitations to our study include the small sample size and considerable coefficient of variability.”

James Longstreth, Ph.D.
Longstreth & Associates, Inc.
Mundelein, Illinois

Sally Faith Dorfman, M.D., MSHSA
Vladimir Yankov, M.D.
Ferring Pharmaceuticals Inc.
Parsippany, New Jersey

Reference
1. Blake E, Norris P, Dorfman S, Longstreth J, Yankov V. Single and multidose pharmacokinetic study of a vaginal micronized progesterone insert (Endometrin®) compared to vaginal gel in healthy reproductive-aged female subjects. Fertil Steril in press.

To the Editor:

We have read with great interest the study by Singer et al. (1) and we would like to comment on the relationship between anti-müllerian hormone (AMH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels in polycystic ovary syndrome (PCOS).

Singer et al., in women undergoing IVF, reported a negative association between FSH and AMH serum levels, concluding that AMH levels are highly predictive of FSH levels and can be used as independent indicators of ovarian reserve (1).

Recently, Dumesic et al. also reported that intrafollicular AMH levels negatively correlated with FSH in follicles of normoandrogenic ovulatory women undergoing IVF, concluding that intrafollicular AMH reflects follicle sensitivity to FSH (2).

In an earlier study (3), we have noted increased serum AMH levels in PCOS women. Moreover, AMH levels were negatively correlated to body mass index (BMI) and were independently predicted by LH, testosterone and BMI. We concluded that increased LH levels might be an independent link between PCOS-associated disorders of ovulation and the increased serum AMH concentrations.

In order to assess whether the findings of Singer et al. and Dumesic et al. from a cohort of older women undergoing IVF, could be applied to younger women with PCOS, we selected a population of 50 ovulatory women as controls and 200 PCOS women with biochemical hyperandrogenemia, oligo- and/or anovulation (ANOV), and/or polycystic ovaries on ultrasound (PCO).

Serum AMH levels were not correlated with serum FSH levels in PCOS and ovulatory women. On the contrary, serum AMH levels were correlated to LH levels in PCOS women (r=0.414, p<0.001). Serum AMH levels were also negatively correlated to BMI for both PCOS (r=-0.222, p=0.007) and ovulatory women (r=-0.696, p<0.001). Indeed, by dividing all women according to BMI (limit 25 Kg/m²), serum AMH levels were significantly higher in normal weight PCOS (6.88±3.60 ng/mL vs. 4.99±2.16 ng/mL, p<0.001) and normal weight ovulatory women (4.11±1.29 ng/mL vs. 2.41±0.25 ng/mL, p<0.001).

We can therefore raise the hypothesis that serum AMH levels are differently modulated in conditions of pure ovarian reserve, like women undergoing IVF, compared to young women with hyperandrogenemia. The latter is particularly true for normal-weight hyperandrogenic PCOS women, which actually represent the more “pure” form of PCOS.

In conditions of increased LH and normal-to-low FSH, such as PCOS, AMH serum levels are increased and tend to be associated to serum LH, while in conditions of increased FSH such as premature ovarian failure, AMH serum levels are decreased and tend to be associated to serum FSH.

Neoklis A. Georgopoulos, Ph.D.^a
Athanasia Piouka, M.Sc.^b
Ilias Katsikis, Ph.D.^b
Alexandros D. Saltamavros, Ph.D.^a
George Decavalas Ph.D.^a
Dimitrios Panidis, Ph.D.^b
aDepartment of Obstetrics and Gynecology
Division of Reproductive Endocrinology
University of Patras Medical School
Patras, Greece
^bDivision of Endocrinology and Human Reproduction
Second Department of Obstetrics and Gynecology
Aristotle University of Thessaloniki
Thessaloniki, Greece

References
1. Singer T, Barad DH, Weghofer A, Gleicher N. Correlation of antimüllerian hormone and baseline follicle-stimulating hormone levels. Fertil Steril 2009;91:2616-9.

2. Dumesic DA, Lesnick TG, Stassart JP, Ball GD, Wong A, Abbott DH. Intrafollicular antimullerian hormone levels predict follicle responsiveness to follicle-stimulating hormone (FSH) in normoandrogenic ovulatory women undergoing gonadotropin releasing-hormone analog/recombinant human FSH therapy for in vitro fertilization and embryo transfer. Fertil Steril 2009;92:217-21.

3. Piouka A, Farmakiotis D, Katsikis I, Macut D, Gerou S, Panidis D. Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: relationship with increased luteinizing hormone levels. Am J Physiol Endocrinol Metab 2009;296:E238-43.

The Authors Respond:

We appreciate the interest of Dr. Georgopoulos and colleagues in our study. In response to our observation of an inverse relationship between FSH and AMH among women undergoing in vitro fertilization, they have offered their own observation that among women with polycystic ovarian syndrome the FSH/AMH relationship is not present. They further offer evidence that AMH concentration is directly related to luteinizing hormone (LH) among women with PCOS.

While a relationship between serum AMH and LH concentration among women with PCO is plausible, we fail to see the utility of such an observation. AMH is produced by antral and pre-antral follicles. Antral follicles decrease with reproductive aging, and as a consequence, serum concentration of AMH falls (1). Fewer antral follicles result in a fall in both inhibin and sex steroids, leading to a consequent rise in FSH.(2) Thus, while serum AMH is directly linked to ovarian reserve, serum FSH is a few steps removed from this process.

Our study related the serum FSH to serum AMH.(3) Such a relationship may be used to help clinical interpretation of AMH concentration with regard to prediction for ovarian reserve. We did not imply a direct physiologic relationship between AMH and FSH secretion, other than the common link as a reflection of follicular dynamics.

The observation of increased AMH among women with PCO-like ovaries is a further example of the direct relationship of serum AMH to antral follicles, which are abundantly present in the ovaries of women with PCOS. It is well known, and was at one time part of the diagnostic criteria for PCOS, that women with this condition often have increased serum concentration of LH. Thus a direct relationship of LH and AMH demonstrated in a population of women with PCOS is not a surprise. Indeed, elevated serum concentration of AMH may have utility in the diagnosis of PCOS(4), not as a surrogate for serum LH measurement, but as a direct reflection of the increased antral follicle pool.

We did not propose that AMH levels are modulated by FSH and do not believe that our correspondents have offered credible evidence to support their hypothesis that BMI and LH directly modulate AMH among women with PCO. Instead we interpret both our study and the data provided by Georgopoulos et al. as further support of the well established relationship between antral follicle numbers and serum AMH.

David H Barad, M.D., M.S.
Norbert Gleicher, M.D.
Tomer Singer, M.D.
The Center for Human Reproduction
New York, New York

References
1. Fanchin R, Schonauer LM, Righini C, Guibourdenche J, Frydman R, Taieb J. Serum anti-Mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum Reprod 2003;18:323-7.

2. Scheffer GJ, Broekmans FJ, Dorland M, Habbema JD, Looman CW, te Velde ER. Antral follicle counts by transvaginal ultrasonography are related to age in women with proven natural fertility. Fertil Steril 1999;72:845-51.

3. Singer T, Barad DH, Weghofer A, Gleicher N. Correlation of antimullerian hormone and baseline follicle-stimulating hormone levels. Fertil Steril 2009;91:2616-9.

4. Piltonen T, Morin-Papunen L, Koivunen R, Perheentupa A, Ruokonen A, Tapanainen JS. Serum anti-Mullerian hormone levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. Hum Reprod 2005;20:1820-6.

To the Editor:

I have read with interest the case report by Azem et al. (1), and congratulate them. However, several points need clarification:

1. The authors conclude from the presence of germinal vesicle–stage oocytes in the medium on the presence of antral follicles. Antral follicles can be clearly visualized by ultrasound and seen in the extirpated ovary. The authors failed to mention the visualization of antral follicles in vivo, by ultrasound, or in vitro in the excised ovary. Indeed, in the same issue of Fertility and Sterility, Revel et al. (2), report aspirating 179 oocytes in all observed follicles before ovarian cryopreservation in patients aged 5-20 years. The finding of immature ova in the medium is not a proof of antral follicles. It could have been mechanically dispersed from preantral follicles.

2. The authors refer to the Human Reproduction Update editorial (3) in response to our (4) and our colleagues’ (5)reviews. The editorial mentioned a prospective randomized study that has been presented as a poster, claiming that if published, after peer review, it may settle the debate. Indeed, the referred study has been published in Fertility and Sterility (6), showing 11.4% premature ovarian failure (POF) in the GnRH-a group vs. 66% POF in controls (P<.001). In the last decade, 16 publications had similar positive results (4–11). A recent ASCO publication (9), similarly found the rates of POF to be 0 – 6% with GnRH-a vs. 32% – 47% without it. Recently, another prospective randomized study (10) had similar results. Thus, there are three prospective peer reviewed studies (6,10,11) with positive results. A recent meta-analysis (12) has found a significant beneficial role for the agonists in both preservation of ovarian function and conception: 68% increase in the rate of preserved ovarian function (RR = 1.68, CI=1.34-2.1). Among the GnRH-a treated women, 22% achieved pregnancy compared with 14% without GnRH-a (RR = 1.65, CI=1.03-2.6) (12). This meta-analysis (12) concluded that women facing chemotherapy should be counseled about ovarian preservation options, including the use of GnRH-a.

3. Indeed there are many unknown and equivocal matters in this important issue. Similarly, the group that published the first delivery after transplantation of ovarian tissue has recently published their disappointing results: "IVF in women with orthotopically grafted frozen-thawed ovarian tissue involves a higher risk of empty follicles, abnormal or immature oocytes, and low embryo transfer rates." (13). Therefore, we should all admit we are still far from having a ubiquitous solution for all these young patients.

4. Azem et al. (1) have matured in vitro the ova retrieved from the medium and cryopreserved for future IVF. However, it has been shown that ova exposed to chemotherapy exhibit a high rate of chromosomal aberrations, miscarriages and possibly fetal malformations (14). Chemotherapy is mutagenic to germ cells at various stages of maturation (14).

5. Ovarian cryopreservation, GnRH-a administration, and follicular aspiration should be offered to all such patients. Furthermore, GnRH-a can effectively prevent the thrombocytopenia-associated menorrhagia in these patients (15).

Zeev Blumenfeld, M.D.
Reproductive Endocrinology
Department of Obstetrics and Gynecology
Rambam Medical Center
Technion-Faculty of Medicine
Haifa, Israel

References
1. Azem F, Hasson J, Cohen T, Shwartz T, Mey-Raz N, Almog B, et al. Retrieval of immature oocytes after chemotherapy for Hodgkin’s disease and prolonged ovarian down-regulation with gonadotropin-releasing hormone agonist. Fertil Steril. 2009; 92: 828.e1–e2.

2. Revel A, Revel-Vilk S, Aizenman E, Porat-Katz A, Safran A, Ben-Meir A, et al. At what age can human oocytes be obtained? Fertil Steril. 2009; 92: 458-63.

3. Hughes EG, Neal MS. Ovarian suppression to protect against chemotherapy- induced ovarian toxicity: helpful or just hopeful. Hum Reprod Update 2008;14:541–2.

4. Blumenfeld Z, von Wolff M. GnRH-analogues and oral contraceptives for fertility preservation in women during chemotherapy. Hum Reprod Update 2008;14:543–52.

5. Beck-Fruchter R, Weiss A, Shalev E. GnRH agonist therapy as ovarian protectants in female patients undergoing chemotherapy: a review of the clinical data. Hum Reprod Update 2008;14:553–61.

6. Badawy A, Elnashar A, El-Ashry M, Shahat M. Gonadotropin-releasing hormone agonists for prevention of chemotherapy-induced ovarian damage: prospective randomized study. Fertil Steril. 2009; 91: 694-7.

7. Blumenfeld Z, Avivi I, Eckman A, Epelbaum R, Rowe JM, Dann EJ. Gonadotropin-releasing hormone agonist decreases chemotherapy-induced gonadotoxicity and premature ovarian failure in young female patients with Hodgkin lymphoma. Fertil Steril 2008; 89: 166-73.

8. Blumenfeld Z. How to preserve fertility in young women exposed to chemotherapy? The role of GnRH agonist cotreatment in addition to cryopreservation of embrya, oocytes, or ovaries. Oncologist. 2007;12: 1044-54. Review.

9. Moore HC. Ovarian failure after chemotherapy for breast cancer and the role of gonadotropin-releasing hormone analogs in protection of ovarian function. In: American Society of Clinical Oncology 2008 educational book. Alexandria (VA): American Society of Clinical Oncology, 2008: 39–42.

10. Sverrisdottir A, Nystedt M, Johansson H, Fornander T. Adjuvant goserelin and ovarian preservation in chemotherapy treated patients with early breast cancer: results from a randomized trial. Breast Cancer Res Treat. Published online January 20, 2009.

11. Ataya K, Rao LV, Laurence E, Kimmel R. Luteinizing hormone-releasing agonist inhibits cyclophosphamide induced ovarian follicular depletion in Rhesus monkeys. Biol Reprod 1995; 52:365–72.

12. Clowse ME, Behera MA, Anders CK, et al. Ovarian preservation by GnRH agonists during chemotherapy: a meta-analysis. J Womens’ Health (Larchmt). 2009; 18: 311-9.

13. Dolmans MM, Donnez J, Camboni A, Demylle D, Amorim C, Van Langendonckt A, Pirard C. IVF outcome in patients with orthotopically transplanted ovarian tissue. Hum Reprod. 2009 Aug 11. [Epub ahead of print].

14. Meirow D, Schiff E. Appraisal of chemotherapy effects on reproductive outcome according to animal studies and clinical data. J Natl Cancer Inst Monogr. 2005;34:21-5.

15. Meirow D, Rabinovici J, Katz D, Or R, Shufaro Y, Ben-Yehuda D. Prevention of severe menorrhagia in oncology patients with treatment-induced thrombocytopenia by luteinizing hormone–releasing hormone agonist and depo-medroxyprogesterone acetate. Cancer 2006; 107: 1634–41.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.09.037

The Authors Respond:

The biological plausibility of using GnRH agonists for fertility preservation is a matter of controversy (1). Several possible explanations for the beneficial effect of GnRH agonists to minimize chemotherapy-associated gonadotoxicity have been suggested (1, 2). One of them proposes that the hypogonadotropic milieu decreases the number of primordial follicles that enter the differentiation stage, which are more vulnerable to chemotherapy than arrested follicles. If this were the case, even preantral follicles (as suggested by Dr. Blumenfeld) would not be expected to be found in the medium.

There is no argument about the importance of the prospective randomized study by Badawy et al., but it had not been given approval by an ethical committee (3). At any rate, it was not our intention to join in the debate between the different studies mentioned by Dr. Blumenfeld: we believe that these issues have already been dealt with comprehensively in the paper by Oktay et al. (1)

We respect the works that Dr. Blumenfeld cited in support of his stance but, at the same time, wish to point out that he did not refer to the letter to the editor written by his own colleagues, which refuted his findings (4).

We are aware of the mutagenic potential of chemotherapy. In the reported case, however, our patient was treated by ABVD, which is well known for its low mutagenic effect. Additionally, if Dr. Blumenfeld’s assumption is correct, the follicles should be initially quiescent and so would not be affected by chemotherapy which mainly affects cleaving cells.

Foad Azem, M.D.
Dalit Ben-Yosef, Ph.D.
Beni Almog, MD
Joseph B. Lessing, M.D.
Ami Amit, M.D.

Tel Aviv Sourasky Medical Center
Tel Aviv, Israel

References
1. Oktay K, Sönmezer M, Oktem O, Fox K, Emons G, Bang H. Absence of conclusive evidence for the safety and efficacy of gonadotropin-releasing hormone analogue treatment in protecting against chemotherapy-induced gonadal injury. Oncologist 2007; 12:1055-66.

2. Muller U, Stahel A. Gonadal function after MA COP-B or VACOP-B with or without dose intensification and ABMT in young patients with aggressive non-Hodgkin’s lymphoma. Ann Oncol 1993;4:399–402.

3. Badawy A, Elnashar A, El-Ashry M, Shahat M. Gonadotropin-releasing hormone agonists for prevention of chemotherapy-induced ovarian damage: prospective randomized study. Fertil Steril 2009; 91:694-7.

4. Kol S, Leithman A, Itskovitz-Eldor J. Evidence-based medicine or just a theory? Fertil Steril 2009; 92:e9.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.09.038

To the Editor:

We read the article by Tsoumpou et al. (1) with interest and commend the authors’ attempt to address this topical issue. The decision on what to do with regard to endometriomas in women having IVF remains an everyday challenge. The authors have done well to summarize the available data addressing this issue.

Their article clearly highlights the paucity of good quality evidence in managing endometrioma in women embarking on IVF, and here-in lies the problem with this systematic review. The validity of the results of a systematic review is only as good as the quality of the studies reviewed. A good meta-analysis of data derived from design-weak studies obviously results in conclusions based on weak evidence. It is for this reason that experts propose that only methodologically sound studies should be included in a meta-analysis. Apart from the obvious non-randomized nature of the studies included in this meta-analysis, the population in the various studies were in no way similar.

Following the systematic review, the authors conclude that “available data suggest surgical management of endometriomas has no significant effect on IVF pregnancy rates and ovarian response to stimulation compared with no treatment.” We wonder how this conclusion, which is based on very weak evidence, will impact on the clinical decisions of the readers. We agree with the authors that randomized controlled trials are needed to guide clinical decisions in these cases. Until such a time, treatment should be individualized and the decision to proceed to surgery or not before IVF must be made on a case-by-case basis.

Various factors will influence clinical decisions. These include (but are by no means limited to) size of the cyst, previous ovarian surgery, previous ovarian response to stimulation/IVF outcome and, not the least, the patient’s choice. It is important that in counseling the patients we do make it clear that there is no proven correct answer in these cases, as the evidence is sparse. Several assumptions including the purported adverse effect of the cyst content on oocyte quality as alluded by the authors in their discussion have little or no evidence to support them.

Finally, the authors have suggested an easy and seemingly logical flow chart for the management of endometrioma in women undergoing IVF. This also is not based on any evidence, and as such patients should not be treated in boxes. We strongly agree with the authors that best practice recommendations on this topic must await randomized controlled trials. Until then, each case must be treated on its own merits.

Vivek Nama, MRCOG^a
Emmanuel Kalu, MRCOG^b

^bAssisted Conception Unit
Queen Mary’s Hospital
London, United Kingdom

Reference
1. Tsoumpou I, Kyrgiou M, Gelbaya TA, Nardo LG. The effect of surgical treatment for endometrioma on in vitro fertilization outcomes: a systematic review and meta-analysis. Fertil Steril. 2009;92(1):75-87.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.09.003

The Authors Respond:

We would like to thank Drs. Nama and Kalu for their interest in our recent manuscript.

A systematic review aims to collect, review, present and critically appraise all available evidence regardless of how weak or strong that might be. We believe that we have clearly accomplished that in this paper.

As far as the meta-analysis is concerned we agree entirely that the quality of a meta-analytical pooling depends on the quality of the included trials. To date the relevant available data come from retrospective, case-controls and prospective cohort studies. We do not agree that they should be dismissed from inclusion as non-methodologically sound. The decision to include these studies in our review and meta-analysis was based on the fact that they addressed the same clinical question, and neither the clinical nor the methodological heterogeneity amongst them appeared to alter the treatment effect.

The authors refer to the conclusions in the abstract emphasizing that no definite conclusion can be drawn from the available evidence. We were cautious indeed to mention that randomized controlled trials (RCTs) are needed before producing best-practice recommendations on this topic. Further, as stated in the “Conclusions” section of the manuscript, standard management of endometrioma in subfertile women undergoing IVF remains controversial, owing to the insufficient evidence to suggest superiority of one treatment strategy over another. We also stated that until a large, properly designed RCT is conducted and definite conclusions presented, the management of endometrioma before IVF should be individualized. In the last paragraph of our discussion we stated that, based on the available evidence including this systematic review we were far from reaching any robust recommendations for routine clinical practice.

Regarding the flow chart, we aimed to propose what could be seen as at-a-glance guidance for the management of subfertile women with endometriomas needing IVF treatment. The arbitrary cut-off we proposed was based on our clinical experience and is far from being considered as evidence-based guidelines.

Ioanna Tsoumpou, M.B. Ch.B.
Tarek A. Gelbaya, M.D.
Luciano G. Nardo, M.D.

Department of Reproductive Medicine
St Mary’s Hospital
Manchester, United Kingdom

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.09.004

To the Editor:

In a recent article in Fertility and Sterility, Fedele et al. discuss the efficacy of the Williams vaginoplasty as a method of treatment for the colpopoiesis in Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome patients with a pelvic kidney, and compare the safety and efficacy of this method with those of the Vecchietti’s laparoscopic modification. (1)

According to authors’ opinion, expressed in the Discussion section of the above mentioned article: “The extraperitoneal approach implied in the Williams techniques certainly minimizes the risks associated with the presence of a pelvic kidney. This vaginoplasty technique also has the advantage of having an extraperitoneal approach and being a relatively simple procedure.”

It is true that, due to the nature of the procedure, Williams vaginoplasty is indeed the safest method to perform, for the creation of a neovagina, especially in this subgroup of MRKH patients, as no dissection of the vesicorectal space is required.

Unfortunately, we are not in position to agree with authors’ further statement that “the presence of perineal hair and the nonphysiologic external angle of the neovagina that results from this technique do not allow optimal anatomical results.”

According to our experience, published several times in the past (2-4), after the performance of the Williams vaginoplasty, the functional axis of the neovagina tends to be very similar to the anatomical deviation of a normal vagina.

Furthermore, we would like to assure you that despite the fact that initially we were also surprised, during the long-term follow up of 200 cases treated by the Creatsas modification of the Williams vaginoplasty, in a course of more than 20 years, we find no perineal hair present at the inner segment of the neovagina.

In conclusion, we strongly believe that for the creation of a neovagina, especially in case of contemporaneous existence of a pelvic kidney, the simplest, quickest, most effective, but beyond all safest method, is the Creatsas modification of Williams vaginoplasty.

George Creatsas, MD, PhD, FACS, FRCOG, FACOG
Panagiotis Christopoulos, MD, MSc, PhD
Division of Pediatric-Adolescent Gynecology and Reconstructive Surgery
2nd Department of Obstetrics and Gynecology
University of Athens Medical School
“Aretaieio” Hospital
Athens, Greece

References
1. Fedele L, Frontino G, Motta F, Restelli E, Candiani M. Creation of a neovagina in Rokitansky patients with a pelvic kidney: comparison of long-term results of the modified Vecchietti and McIndoe techniques. Fertil Steril. 2009 Jan 24. [Epub ahead of print]

2. Creatsas G, Deligeoroglou E. Expert opinion: vaginal aplasia: creation of a neovagina following the Creatsas vaginoplasty. Eur J Obstet Gynecol Reprod Biol. 2007 Apr;131(2):248-52.

3. Botsis D, Deligeoroglou E, Christopoulos P, Aravantinos L, Papagianni V, Creatsas G. Ultrasound imaging to evaluate Creatsas vaginoplasty. Int J Gynaecol Obstet. 2005 Apr;89(1):31-4.

4.Creatsas G, Deligeoroglou E, Makrakis E, Kontoravdis A, Papadimitriou L. Creation of a neovagina following Williams vaginoplasty and the Creatsas modification in 111 patients with Mayer-Rokitansky-Küster-Hauser syndrome. Fertil Steril. 2001 Nov;76(5):1036-40.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.08.047

The Authors Respond:

We apologize to Drs. Creatsas and Christopoulos for not having specified more in detail the characteristics of the Williams vulvovaginoplasty in the Discussion section of our paper entitled “Creation of a neovagina in Rokitansky patients with a pelvic kidney: comparison of long-term results of the modified Vecchietti and McIndoe techniques.” (1)

As we personally do not have experience in the Williams surgical procedure, the comments we have reported in our study had been obtained from previous studies regarding this technique (2-5). Undoubtedly, one of the primary factors of success is the surgeon’s experience in the surgical technique and in the postoperative management, both of which may have yielded the optimal results described in the publications of Creatsas and co-workers.

Luigi Fedele, MD
Giada Frontino, MD
Francesca Motta, MD
Elisa Restelli, MD
Massimo Candiani, MD
Clinica Ostetrica e Ginecologica I
Universita di Milan Fondazione Policlinico
Mangiagalli e Regina Elena
Milan, Italy

References
1. Fedele L, Frontino G, Motta F, Restelli E, Candiani M. Creation of a neovagina in Rokitansky patients with a pelvic kidney: comparison of long-term results of the modified Vecchietti and McIndoe techniques. Fertil Steril. 2009, in press.

2. Miller RJ and Breech LL. Surgical correction of vaginal anomalies. Clinical Obstetrics and Gynecology 2008;51(2):223-6.

3. Edmonds KD. Congenital malformations of the genital tract and their management. Best Pract Res Clin Obstet Gynecol 2003;17(1):19-40.

4. Noguchi S, Nakatsuka M, Sugiyama Y, Chekir C, Kamada Y, Hiramatsu Y. Use of artificial dermis and recombinant basic fibroblast growth factor for creating a neovagina in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome. Hum Reprod. 2004;19(7):1629-32.

5. Dewhurst’s Textbook of Obstetrics and Gynaecology, 7th Ed., Wiley-Blackwell Publishers, 2007.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2009.08.048