References Belen Martinez-Madrid

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					Letters to the editor

References                                                                      ovary and the entire ovary cryopreserved. Should a cryovial
Bedaiwy MA, Falcone T, Biscotti C et al. Cryopreservation of intact human       with an adequate size be available, the rest of the process
  ovary with its vascular pedicle. Hum Reprod 2004;19:i77 (O-220).              could have been performed with the ovary intact as we did in
Bedaiwy MA, Hussein MR, Biscotti C, Falcone T. Cryopreservation of intact       our initial experiment in sheep ovaries (Bedaiwy et al.,
  human ovary with its vascular pedicle. Hum Reprod 2006;21:3258–9.
Donnez J, Martinez-Madrid B, Jadou Pl et al. Ovarian tissue cryopreservation
                                                                                2003). A specially designed cryovial and cryochamber to
  and transplantation: a review. Hum Reprod Update 2006;12:519–35.              accommodate an entire ovary with its vascular pedicle is
Kupesic S, Kurjak A, Bjelos D et al. Three-dimensional ultrasonographic         being developed by industry.
  ovarian measurements and in vitro fertilization outcome are related to age.      Second, both ovaries fit after bisection in the cryovial
  Fertil Steril 2003;79:190–7.
Martinez-Madrid B, Dolmans MM, Van Langendonckt A et al. Freeze-thawing
                                                                                described in our study. We agree with their concern regarding
  intact human ovary with its vascular pedicle with a passive cooling device.   the fact that fitting ovaries with larger sizes in this particular
  Fertil Steril 2004;82:1390–94.                                                cryovial is challenging. In this limited series, we purposely
Motta PM, Balboni GC. Apparato genitale femminile. In: Ermes (ed.)
  Anatomia Umana, 3rd edn. Milano, Italy: Ed. Ermes, 1994.
                                                                                chose older women whom we knew would have small
                                                                                ovaries. The data they quoted in their letter state that there
                        Belen Martinez-Madrid and Jacques Donnez1               is a progressive decline in ovarian volume from 10 ml in
                                                                                women less than 30 years of age to 6.8 ml in women from
Department of Gynecology, Universite Catholique de Louvain,                     36 to 40 years of age. Our two patients were 44 and 46 years
                                         Brussels, Belgium                      of age and easily fit into the 5 ml cryovial. We agree that in
1                                                                               clinical practice when we are dealing with young women we

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 Correspondence address. Department of Gynecology,
Universite Catholique de Louvain, Brussels, Belgium.                            can never use the present cryovials. We simply chose ovaries
E-mail:                                                   that we knew were at the end of their reproductive life with
                                                                                small ovaries. As for the preparation of the ovarian cortical
                                     doi:10.1093/humrep/dem047                  strips, we used the protocol described by Gosden and associates
                        Advance Access publication March 30, 2007               (Gosden et al., 1994).
                                                                                   Third, we used fetal calf serum for experimentation purposes
                                                                                only. Indeed animal free media would be the only way to
                                                                                proceed if autotransplantation is planned. This study was
                                                                                again just an experiment to assess feasibility.
Reply: Cryopreservation of intact human ovary with its                             Fourth, this paper was presented, in part, as an abstract in
vascular pedicle—or cryopreservation of hemi ovaries                            the ESHRE meeting of 2004. Only one patient from that
                                                                                abstract published in 2004 was included in this report and
Sir,                                                                            the other patient and all the controls in the current manuscript
We would like to thank Professor Donnez and associates for                      were from subsequent work. The patient in common between
their interest in our manuscript entitled ‘Cryopreservation of                  the abstract and the current manuscript is the 44 years old
intact human ovary with its vascular pedicle’. Regarding the                    patient who underwent laparoscopic hysterectomy and bilat-
questions they raised with respect to our work, our responses                   eral salpingo-ophrectomy. The other patient from the 2004
are as follows.                                                                 ESHRE abstract, who underwent cryopreservation of both
   First, we need to re-emphasize that this work is purely experi-              ovaries as ovarian cortical strips, was not included in the
mental and the result of the observation that ischemia is                       current manuscript because; (i) both ovaries were cryopre-
responsible for most of the graft failures. This work is simply                 served as ovarian cortical strips and we did not do a whole
an evolution of our sheep data published previously. We first                    ovary freezing on her and (ii) we did not run some of the in
described the technique of cryopreserving a whole ovary                         vitro assessment strategies, namely, the CD 34, PAS and
with its vascular pedicle in the sheep animal model, and we                     Masson’s trichome stains on the tissues obtained from this
demonstrated the potential feasibility as evidenced by vascular                 patient.
patency and hormonal function (Jeremias et al., 2002; Bedaiwy                      Fifth, as demonstrated in the elaborate in vitro experiments
et al., 2003). More recently, Imhof et al. (2006) described preg-               we did, adequate and comparable tissue survival was
nancy and delivery using an orthotopic approach of whole                        achieved in both study arms using the described protocol of
ovary vascular anastomosis (Imhof et al., 2006). Our present                    thawing. Cannulation of the largest branch of the ovarian
paper was intended to take it to the next experimental level                    vessels in the section that did have the pedicle connected was
with human ovaries (Bedaiwy et al., 2006). We did not intend                    performed to wash out the cryoprotectants. We also would
to proceed to transplantation but simply to see if we can carry                 like to emphasize that we did not use sucrose and PBS as a
out the steps we reported.                                                      thawing medium in this study and it was quoted in the 2004
   As noted in our manuscript, the main and the most                            ESHRE abstract by mistake. We used only leibovitz L-15
critical step in the entire process—the perfusion of the                        medium supplemented with 10% FCS to wash out the cryopro-
cryoprotectant—was performed via the ovarian vessels to                         tectant following the protocol we used earlier (Bedaiwy et al.,
ensure adequate distribution of the cryoprotectant via the                      2003).
intraovarian vascular network. Bisecting the ovary to fit in                        Sixth, regarding our statement that ovarian tissue could tole-
the largest available cryovial thereafter would not change the                  rate adequately varying concentrations of DMSO with compar-
fact that the perfusion process was performed on an intact                      able post-thaw survival, we agree that the difference in
                                                                                                                                  Letters to the editor

concentration was not large, we used 1.5 M DMSO and they                            The current study was designed to show superiority of HP
used 1.4 M DMSO (Martinez-Madrid et al., 2004).                                  hMG over rFSH (Gonal F) based on the retrospective finding
   In conclusion, we understand the controversy in this area as                  from a previous comparative study (The EISG study) (The
cryopreservation of an intact organ is really challenging and                    European and Israeli Study Group, 2002, Platteau et al.,
still in its infancy. We wish to again point out that this work                  2004) that found HP hMG might be associated with improved
was only an extension of our whole organ work in sheep                           outcomes in the IVF population. In the MERIT trial, all efforts
and in no way was intended for clinical use. We congratulate                     were made to rigorously test the hypothesis from the EISG
this group for successfully carrying out cryopreservation of                     study while maintaining the design of the EISG study.
a whole ovary with a vascular pedicle using the vascular                         Despite efforts to showcase the potential benefits of LH
pedicle for perfusion of cryoprotectant in nine patients. We                     add-back, the MERIT trial failed to demonstrate any superior-
look forward to seeing the results published and look forward                    ity of HP hMG over recFSH. Instead, it confirmed non-
to their first successful transplant.                                             inferiority as pertaining to pregnancy outcome. Two particular
                                                                                 choices in protocol design are worthy of comment.
                                                                                    In the MERIT trial (Andersen et al., 2006), all patients
                                                                                 received a long GnRH agonist down-regulation protocol
Bedaiwy MA, Hussein MR, Biscotti C et al. Cryopreservation of intact human       (0.1 mg triptorelin/day) prior to initiation of ovarian stimu-
   ovary with its vascular pedicle. Hum Reprod 2006;21:3258–69.
Bedaiwy MA, Jeremias E, Gurunluoglu R et al. Restoration of ovarian function     lation. Interestingly, in a Ferring-sponsored study published
   after autotransplantation of intact frozen-thawed sheep ovaries with          in 2000, Janssens et al. (2000), found that doses of

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   microvascular anastomosis. Fertil Steril 2003;79:594– 602.                    triptorelin as low as 15 mg were sufficient to suppress the LH
Gosden RG, Baird DT, Wade JC et al. Restoration of fertility to
   oophorectomized sheep by ovarian autografts stored at 2196 degrees C.
                                                                                 surge, whereas 50 mg of triptorelin was as effective as
   Hum Reprod 1994;9:597–603.                                                    100 mg of triptorelin. Furthermore, it has previously been
Imhof M, Bergmeister H, Lipovac M et al. Orthotopic microvascular                shown that triptorelin is one of the more potent GnRH agonists
   reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy     (Devroey et al., 1994). Therefore, the choice of 100 mg/day
   and live birth. Fertil Steril 2006;85:1208–15.
Jeremias E, Bedaiwy MA, Gurunluoglu R et al. Heterotopic autotransplantation     triptorelin would be expected to accentuate any potential
   and vascular anastomosis: a novel surgical technique. Fert Steril             benefit of LH add-back during subsequent stimulation with
   2002;77:127.                                                                  HP hMG.
Martinez-Madrid B, Dolmans MM, Van Langendonckt A et al. Freeze-thawing
   intact human ovary with its vascular pedicle with a passive cooling device.
                                                                                    Despite focusing on IVF-patients only, the MERIT trial did
   Fertil Steril 2004;82:1390– 4.                                                not demonstrate outcomes significantly different from the
                                                                                 EISG study (Andersen et al., 2006). Thus it appears that focus-
                   Mohamed A.Bedaiwy1 and Tommaso Falcone                        ing on the IVF population does not indicate that LH add-back is
                                                                                 mandatory and the question of which subgroup might benefit
    Department of Gynecology and Obstetrics, Cleveland Clinic                    from LH add-back remains unanswered. Perhaps it would be
                               Foundation, Cleveland, Ohio,                      more logical to prevent the iatrogenic depletion of LH-activity
                                                        USA                      by simply reducing the daily GnRH agonist dose currently
 To whom correspondence should be addressed at: Department                       employed in routine IVF practice?
of Gynecology and Obstetrics, Cleveland Clinic Foundation,                          I would also like to address the choice of the starting dose of
Cleveland, Oh, USA.                                                              225 IU for rFSH in standard patients. We speculate that for a
E-mail:                                                     large proportion of these women, who have relatively good
                                                                                 prognoses, this dose could be considered inappropriately
                                  doi:10.1093/humrep/dem048                      high. Yet the authors fail to explain the rationale for choosing
                     Advance Access publication March 30, 2007                   such a high dose. Indeed, a substantial body of work has been
                                                                                 published in which 150 IU/day was used as the standard dose
                                                                                 for standard patients (Latin American Puregon IVF Study
                                                                                 Group, 2001; Popovic-Todorovic et al., 2003). The link
Comparing highly purified hMG and rFSH in patients                                between higher starting doses of rFSH and higher progesterone
undergoing IVF                                                                   levels on the day of hCG administration is well established
                                                                                 (Out et al., 2001; Latin American Puregon IVF study Group,
Sir,                                                                             2001). Thus the use of such a high starting dose is likely to
I read with interest the recent article by Andersen et al., (2006) on            lead to a protocol-driven outcome of more follicles, higher pro-
behalf of the MERIT group reporting clinical outcomes from the                   gesterone levels and a more advanced endometrium. Accord-
randomized, controlled, assessor-blind trial comparing highly                    ingly, it could be argued that some of the pharmacodynamic
purified hMG and recombinant FSH in patients undergoing                           differences alluded to in this trial may be protocol-driven
IVF (Andersen et al., 2006). This was obviously a carefully                      rather than LH-activity derived.
designed trial, and because of the large number of patients                         In addition, the total consumption of gonadotropin in this
studied, the results of the trial and the interpretations thereof                trial was significantly higher in the HP-hMG group, whereas
will carry a great deal of weight within the assisted reproduction               the number of oocytes retrieved was higher in the rFSH
community. It is for this reason that I feel compelled to provide                group. Both findings suggest enhanced efficiency of rFSH
an alternative viewpoint regarding some of the key findings.                      compared with HP hMG.