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1- Uterosacral ligament
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2- Rectum
3- Cardinal ligament
4- Vagina
5- Bladder pillar
6- Urinary bladder
7- Prevesical space
8- Paravesical space
9- Vesicovaginal space
10- Rectovaginal space
11- Pararectal space
12- Retrorectal space
A
B
Figure 6.5. (A) Avascular spaces and their ligamental boundries. (B) Illustrated are the eight avascular spaces
and their anatomic relationship during the laparatomy.
A
B
Figure 6.6. (A) A posterior view of the anterior abdominal wall during the Retzius space development (B) The space of Retzius is developed with
blunt and sharp dissection of fibrofatty tissue. Care is taken to avoid periurethral neurovascular injury.
*
r
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Right
Paravesical
Space
Right obliterated
umbilical artery
Right external
iliac artery
A
Left uterine
artery
Leftt
obliterated
umbilical
artery
Paravesical
Space
*
*
Pararectal
*
Pararectal
Space
C
Space
B
Left external iliac
artery and vein
Obliterated
umbilical artery
Left uterine artery
Left hypogastric
artery
D
Figure 6.7. (A) The right paravesical space isdeveloped. (B) Theparavesicalspaces limited by obturatorinternus muscle and thepelvic diaphragm
laterally, the bladder pillar medially, the endopelvic fascia inferiorly, uterine artery posteriorly, and the medial umbilical ligaments superiorly. (C)
The pararectal spaces are limited by the levator ani laterally, by the rectal pillars medially, by the anterolateral aspect of sacrum posteriorly, and
uterine and cardinal complex anteriorly. (D) The relationship of pararectal and paravaginal spaces with the uterine artery.
Bladder
A
Vesicovaginal
Space
C
*
Ureter and
vesicouterine
ligament
Urete
Cervix
Figure 6.8. (A) The vesicouterine space is a narrow
cul-de-sac between the anterior surface of the uterus
and the cervix and the upper surface of the bladder
when the uterus is in normal anteflexed position.
(B) The vesicouterine and vesicovaginal spaces are
developed. (C) Precise and continuous attention to
the location of the ureter will reduce theincidence of
complications.
B
Sacrouterine
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ligaments
*
Rectouterine-
rectovaginal
Space
Rectum
B
A
Bladder
Right ureter
Left
ureter
C
Figure 6.9. (A) The rectouterine (rectovaginal) space. The entire space, bounded anteriorly by the cervix and by the fornix in the midline, the
uterosacral folds laterally, and the rectum posteriorly. (B) The rectovaginal space is completely developed. (C) Once all ligaments are dissected,
all the spaces open to each other.
Rectum
A
Figure 6.10. (A) Exposure of aortic caval bifurcation. (B) Intraperitoneal view of the bifurcation of the aorta (∗). (Continued)
*
B
80 — Jyoti Yadav, M. Shoma Datta, Ceana Nezhat, Camran Nezhat, and Farr Nezhat
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Left common iliac
artery
C
*
Inferior vena
cava
Right common
iliac artery
Left common
iliac artery
Right common
iliac artery
Left common
iliac vein
D
Middle
Sacral Vein
Left Common
Iliac Vein
Figure 6.10. (Continued )(C) Retroperitoneal view of the bifurcation of the aorta. (D) Anatomic relationships of the bifurcation of the aorta and
inferior vena cava. (E) The middle sacral vessels are in the midline on the sacrum. Care must be taken during dissection of this region.
E
Aorta
r
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Intraperitoneal and Retroperitoneal Anatomy — 81
Inferio
vena
cava
Right
ovarian
vein
D
A
E
B
Aorta
Lumbar
vessels
C
Figure 6.11. (A) The lymph nodes along the right side of the aorta are removed. (B) An anatomical view of the inferior mesenteric artery (IMA)
and its origin from the aorta. (C) Anatomic relationships of the lumbar vessels to the aorta. (D) Anatomic relationships of the drainage of the
right gonadal vein to I.V.C. (E) Anatomic view of the left upper para-aortic area after the lymphadenectomy.
THE URETER
The lumbar ureter lies on the psoas muscle medial to the ovarian
vessels (Figures 6.2D,E, 6.4B, and 6.11A,B). It enters the pelvic
cavity just superficial to the bifurcation of the common iliac artery
and just deep totheovarianvessels, which lie in theinfundibulop-
elvic ligament at the pelvic brim. Itlies in the anterior medial leaf
of the broad ligament as it courses toward the bladder and can
be recognized by its characteristic peristaltic motion. The ureter
then passesjust lateral to the uterosacralligament, approximately
2 cm medial to the ischial spine through the upper part of the
cardinal ligament at the base of thebroad ligament. Here it lies just
82 — Jyoti Yadav, M. Shoma Datta, Ceana Nezhat, Camran Nezhat, and Farr Nezhat
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beneath the uterine artery, approximately 1.5 to 2 cm lateral to
the side of the cervix. The ureter forms a “knee” turn at this point
and travels medially and anteriorly to pass on the anterolateral
aspect ofthe upper third of thevagina toward the bladder (Figure
6.4 A,B and 6.9C).
REFERENCES
1. Nezhat F, Brill AI, Nezhat CH, et al. Laparoscopic appraisal of the
anatomic relationship of the umbilicus to the aortic bifurcation.
J Am Assoc Gynecol Laparosc. 1998;5:135.
SUGGESTED READING
Morrow CP, Curtin JP. Surgical anatomy. In: Gynaecologic Cancer
Surgery. New York: Churchill Livingstone; 1996:67–139.
Nezhat CR, Siegler AM, Nezhat F, Nezhat C, Seidman DS, Luciano
A. Operative Gynecologic Laparoscopy. 2nd ed. New York: McGraw
Hill; 2000.
Pasic RP, Levine RL. A Practical Manual of Laparoscopy: A Clinical
Cookbook. Lancashire, UK: Parthenon Publishing Group; 2002.
Rogers RM Jr, Childers JM. Laparoscopic Gynecologic Anatomy: The
Surgical Essentials.
Smith JR, DelPrioreG, Curtin J,Monaghan JM.AnAtlasof Gynecologic
Oncology. London: Martin Dunitz; 2001.
7 FERTILITY
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Section 7.1. Frontiers in Fertility
Kutluk Oktay and Amr M.A. Azim
The field of reproductive medicine is evolving rapidly. We are living in an erainwhichwhat was seemingly impossibleadecadeago
is being made possible, and century-old dogmas are being challenged. Thanks to new cryopreservation technologies, infertility
and premature ovarian failure, especiallywhen inducedby medical treatments, areno longer unavoidable consequences. Whereas
success with oocyte cryopreservation is gradually approaching
acceptable levels for use in patients who face the risk of ovarian failure due to medical treatments or to create “egg banks”
for oocyte donation, ovarian tissue cryopreservation and transplantation promise to be a way to reverse menopause and restore
fertility. Yet, the recently proposed possibility of the presence of
germ stem cells in human bone marrow is even more intriguing.
Whereas bone marrow and peripheral blood transplants resultin
repopulation of chemotherapy-treated ovaries with primordial
follicles in rodents, germ stem cell markers are already shown in
human bone marrow and peripheral blood.[1] According to this
theory, the ovaries provide signals to the germ stem cells residing
in the bone marrow and recruit new follicles via the bloodstream
“on demand.” Although shocking and contrary to the preexisting dogmathat the ovarian reserve ispredetermined before birth,
this hypothesis is not without supporting evidence inhumansand
nonhuman primates, as illustrated by the following case study.
AN OVARIAN TRANSPLANT CASE
IN SUPPORT OF THE GERM
STEM CELL HYPOTHESIS
One ovary was removed and cryopreserved in a 29-year-old
patient with recurrent Hodgkin’s lymphoma before preconditioning chemotherapy for autologous hematopoietic stem cell
transplantation. The preconditioning chemotherapy containing
a high-dosealkylating agent put the patientin menopause immediately. The patient remained in menopause for 2.5 years, until
her ovarian tissue was thawed and transplanted subcutaneously
to her lower abdominalarea, asdescribed later. Two months after
the transplantation, the patient felt follicle growth underneath her
skin and conceived spontaneously. Three weeks after the termination ofthis abnormal pregnancy, the patientfelt follicle growth
in her graft and had a spontaneous menstruation a week later. In
the next cycle, ovulation was detected by a luteinizing hormone
(LH) surge, contemporaneously with follicle growth in the graft,
at which time the patient had intercourse. The patient conceived
immediately and delivered a healthy female child at term.
Although it is theoretically possible that these pregnancies
following ovarian transplantation could represent a rare event
of ovarian recovery after chemotherapy, occurrence of two preg-
nancies in the 3 months immediately following ovarian transplantation is highly intriguing, especially given the fact that this
patient did not conceive during the previous 2.5 years, when she
appeared to be in menopause.Isitpossible that the chemotherapy
damages the ability of the ovary to recruit germ stem cells from
the bone marrow rather than damaging a preexisting stockpile
of follicles? Could recovery of ovarian function and fertility after
receiving sterilizing chemotherapy be the result of resumption
of de novo production of primordial follicles? Could the transplanted ovary provide signals on behalf of the menopausal ovary
to begin producing primordial follicles or for the bone marrow
to provide germ stem cells to that ovary? Although only future
translational laboratory experiments will determine whether this
mechanism is operational in humans and can be responsible for
ovarian transplant pregnancies, supporting evidence for germ
cell renewal was provided from human and nonhuman primate
studies published more than 50 years ago. Based on histologic
data, Schwarz et al. [2] suggested the continuation of oogenesis
in adult ovaries as early as in 1949. A study by Vermande-Van
Eck [3] put forward a stronger argument for the production of
primordial follicles in nonhuman primate ovaries. In that study,
based on the incidence of atresia and the time it would take for
atretic follicles to be cleared from the ovaries, it was estimated
that the 90% ofovarian reserve would be depletedwithin the first
2 years of life. Considering the fact that the monkeys used in that
study did not evenexperience puberty until the age of 4, and they
remained fertile for 20 years, primordial follicles, neo-oogenesis
remains a real possibility in postnatal ovaries.
The field of reproductive medicine is embracing an exciting future, and in the opinion of the senior author (K.O.), only
by keeping an open mind for alternative explanations for previously dogmatized concepts can we support progress. A perfect
example of this is the evolution, under pioneering leaders such as
Dr. Nezhat, of laparoscopic techniques that were considered
“shocking” a decade ago but are now the standard of care. The
following gives only glimpse of what the future may hold for our
field.
OOCYTE CRYOPRESERVATION
Oocyte cryopreservation has the potential to preserve fertility in
females at risk of losing ovarian function due to medical/surgical
treatments or at constitutionallyhighrisk for early ovarian failure.
This technique may also bypass some of the ethical concerns and
legislative restrictions regarding embryo freezing. An oocyte bank
system could be established to make oocyte donation more practical. Oocyte banking may also become a strategy to circumvent
83
84 — Kutluk Oktay and Amr M.A. Azim
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Table 7.1.1: Slow Freezing of Human Mature Oocytes
No. of Oocytes
Study
Thawed Cumulus Cryoprotectant Survival,% Fertlization,% Cleavage,% IVF/ICSI PR/oocyte,%
Chen (1986, 1988)
[9,13]
van Uem (1997) [14] 4 No DMSO 100 50 — IVF 25
Al-Hasani (1987) [15] 205 Partial DMSO/PROH 25 56/75 — IVF 1
Tucker (1996) [16] 81 No PROH 25 65 100 ICSI 3.7
Porcu (1997, 1998)
[10,17]
Porcu (1998) [18] 1502 No PROH 54 57 91 ICSI 1.1
Borini (1998) [19] 129 No PROH 31 51 94 ICSI 2.3
Tucker (1998) [20] 241 No PROH 31 51 74 ICSI 2.1
Polack de Fried
(1998) [21]
Young (1998) [22] 9 PROH 89 100 62 ICSI 1.1
Winslow (2001) [23] 324 No PROH 68 81 95 ICSI 5.3
Quintans (2002) [24] 109 No PROH 63 59 100 ICSI 5.5
Fosas (2003) [25] 88 No PROH 90 73 — ICSI 4.6
Boldt (2003) [26] 90 No PROH 74 59 85 ICSI 7.8
DMSO, dimethylsulfoxide; PR, pregnancies; PROH, 1,2 propanediol.
age-related decline in oocyte reserve and quality. Oocyte freezing
may also be used if no sperm can be collected or retrieved (e.g.,
nonretrieval of sperm during testicular sperm extraction) during
an in vitro fertilization (IVF) cycle.
The potential problems with oocyte freezing are freeze–
50 Partial DMSO 76 71 60 IVF 6
709 No PROH 56 63 90 ICSI 1.3
10 No PROH 30 66 100 ICSI 0.2
In 1997, Porcu et al. [10] reported the first human live birth after
thawing of mature human oocyte and intracytoplasmic sperm
injection (ICSI). Cha et al. [11] in 1999 reported on a pregnancy
from vitrified immature oocyteand IVFusing 5.5 mol/L ethylene
glycol as a cryoprotectant.
thaw-induced hardening of zona pellucida, cytoskeleton damage, alteration of cortical granules, damage to meiotic spindle,
increased incidence of polyploidy andaneuploidy, and parthenogenetic activation of oocytes. The different variables that may
influence successful oocyte freezing include oocyte quality and
age, maturation stage, presence of cumulus mass, type of cryoprotectant, and cryopreservation protocol (slow freeze–rapid
thaw, vitrification).[4–6] The rate of abnormal fertilization after
oocyte cryopreservation ranges from 5% to 15%. Finally, preimplantation genetic diagnosis (PGD) or at least prenatal determination ofkaryotype is advisable until long-term safety data are
available.
Slow Freezing of Mature Human Oocytes
Oocytes should be frozen shortly after harvesting, about 38 to
40 hours after human chorionic gonadotropin (hCG) injection. Most protocols employ slow freeze–rapid thaw using 1,2propanediol (PROH) and sucrose as cryoprotectants. Sodiumdepleted media were recently introduced.
There is considerable variation in the literature concerning
survival after thawing, fertilization,cleavage, and pregnancyafter
cryopreservation of mature oocytes.
Oocyte cyopreservation is still not as efficient as embryo cryopreservation. Per oocyte live birth rates average around 1.7% to
2.0% compared with approximately 7% with fresh oocytes; this
History
The discovery by Polge, Smith, and Parks [7] in 1948 that fowl
spermatozoa can survive freezing at −70
◦
C in the presence of
corresponds to 20% versus 60% live births per embryo transfer, respectively.[12] Some of the main oocyte cryopreservation
reports are summarized in Table 7.1.1.
glycerol marks the onset of cryopreservation of reproductive cells
and tissues. The first success with oocyte freezing was achieved
in mouse in 1977, followed by other animals. In 1983, Trounson
and Mohr [8] demonstrated that human embryos can be thawed
after freezing with subsequent development and pregnancy. Two
main methods of oocyte freezing have emerged, slow freezing
and vitrification. The first human pregnancy from fertilization
of slow-frozen mature oocyte was reported by Chen [9] in 1986.
Vitrification of Mature Human Oocytes
Vitrification is defined as solidification of solution into a glassy
state without ice formation. High concentrations of cryoprotectants and a very rapid cooling rate are used. The most commonly
used cryoprotectant is ethylene glycol because of its relatively
low toxicity and high permeability through zona and the cellular
Frontiers in Fertility — 85
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Table 7.1.2: Vitrification of Human Mature Oocytes
Survival, Fertilization, Cleavage, PR/oocyte,
Reference No. of Oocytes Cumulus Cryoprotectant % % % IVF/ICSI %
Kuleshova (1999) [28] 17 ? EG/S/straw 65 46% 60 ICSI 5.9
Yoon (2000) [29] 90 Yes EG/S/grid 63 68 89 ICSI 3.3
Chen (2000) [30] 198 No EG/S/straw 91 51 80 ICSI No ET
Katayama (2003) [31] 46 ? EG/DMSO/Cryotop 94 91 90 ICSI 4.3
Yoon (2003) [32] 474 Yes EG/S/grid 69 72 95 ICSI 1.3
EG, ethylene glycol; ET, embryo transfer; S, sucrose.
membrane. Some authorities suggest that vitrification is more
suitable than slow freezing for oocyte cryopreservation because
of control of solute concentration/dehydration, short duration
outside the incubator, brief temperature shock, and preservation
of zona pellucida. Results of vitrification of human oocytes are
presented in Table 7.1.2. Modifications that may improve results
of vitrification include addition of synthetic macromolecules,
microinjection of sugars, use of different cryocontainers (pulled
straws, cryoloops, grids), and cytoskeleton stabilizers (taxanes).
Recently, Liebermann et al. [27] achieved over 80% survival of
1120 mature human oocytes after vitrification using a mixture
of ethylene glycol, dimethylsulfoxide (DMSO), polymer macromolecules, sucrose, and synthetic serum substitute. However,
there have been only10 deliveries from thisprocedure and itstrue
potential remains to be determined. Moreover, because oocytes
are vitrified in an open system, viral cross-contamination in liquid nitrogen is a possibility.
Cryopreservation of Immature Oocytes
Immature human oocytes can survive cryopreservation, with
subsequent maturation to metaphase II oocytes.[33] Chromosomes in prophase oocytes are not aligned alongtheequatorplate,
and they may be theoretically less susceptible to spindle damage
during the freeze–thaw cycle. Immature oocytes can be obtained
during an unstimulated cycle (e.g., during gynecologic surgery)
(Table 7.1.3). Patients with polycystic ovarian disease may particularly benefit from this technology as they tend to produce a
large number of immature oocytes. In addition, freezing immature oocytes has the potential to avoid ovarian hyperstimulation
in patients at risk for ovarian hyperstimulation syndrome or when
increased estrogen levels are considered hazardous (e.g., in breast
cancer patients or patients at risk for thromboembolism). This
method may also help cancer patients who do not have sufficient time to undergo ovarian stimulation before chemotherapy.
Nevertheless, there have been very few reports and live births
with cryopreserved immature oocytes, and the efficiency of this
approach remains to be determined.
IN VITRO MATURATION OF OOCYTES
Retrieving immature oocytes and maturing them in vitro has
the potential to avoid prolonged and expensive controlled ovar-
ian hyperstimulation, ovarian hyperstimulation syndrome, and
exposure to high levels of estrogen when a high-risk condition
exists.
History
Gregory Pincus [41] in 1935 and Roger Edwards [42] in 1965
showed that oocytes may spontaneously resume meiosis when
removedfromantralfollicles.Research in 1970s and 1980s probed
many of the steps involved in oocyte maturation, including the
need for cumulus cells, the importance of follicular and oocyte
size, molecular mechanisms involved in resumption of meiosis,
culture conditions, protein synthesis and gene expression during oocyte maturation, and the effect of priming with folliclestimulating hormone (FSH) and hCG. Although pregnancies
from in vitro maturation (IVM) of immature oocytes obtained
during conventional ovarian stimulation for IVF were reported
in 1983, the first human pregnancy from an immature oocyte
obtained during an unstimulated cycle was reported by Cha et al.
[43] in 1991. In 1994, Trounson et al. [44] reported the first pregnancy after IVM of oocytes from a polycystic ovarian syndrome
(PCOS) patient.
In Vitro Maturation of Oocytes from Antral Follicles
Oocytes forIVM canbe obtained during a cesareansection delivery [43,45], during different phases of the ovarian cycle, from
PCOS patients, and after ovarian stimulation.[44,46,47]
Luteal phase oocytes may display significantly higher maturation ratesthan those obtainedduring follicular phase,although
this has not been a consistent finding in all studies.[48] The presence of a dominant follicle does not affect developmental competence of immature oocytes from antral follicles as they do not
undergo atresia.[49]
Increased age and high day 3 levels of FSH, estradiol
(≥200 pmol/L),and inhibin A(≥10 pg/mL) were associated with
reduced immature oocyte recovery and pregnancy rate.[50]
The ability to resume meiosis after retrieval is largely dependent on follicular and oocyte size. Developmental competence
increases as oocyte diameter increasesfrom 90 to120 μm.[51,52]
Follicles with a minimum diameter of 5 mm can provide
oocytes that can be matured in vitro, irrespective of exposure to
gonadotropin stimulation.[53] Fertilization rate increases progressively in oocytes from follicles 10 mm or greater.
86 — Kutluk Oktay and Amr M.A. Azim
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Table 7.1.3: Freezing of Human Immature Oocytes
No./Cycle/ Survival, Maturation, Fertilization, Cleavage,
Reference Stage Cumulus Protocol % % %; Method % Result
Toth (1994)
[33]
Baka (1995)
∗
[34]
Tucker (1998)
[35]
Son (1996)
[36]
Park (1997)
†
[37]
Cha (2000)
[38]
Wu (2001)
[39]‡
Fuchinoue
(2004) [40]
F, Ficoll (Spectrum Medical Industries); HTF, human tubal fluid; SSS, synthetic serum substitute; GV, germinal vesicle; MI, metaphase I oocyte;
PCOD, polycystic ovarian disease; Stim, stimulated; blast, blastocyst; biochem, biochemical pregnancy; Unstim, unstimulated.
∗
Oocytes matured before freezing had significant spindle abnormalities.
†High incidence of chromosomal abnormalities in oocytes thawed. No attempt at fertilization.
‡Oocytes aspirated from 4- to 11-mm follicles during chocolate cyst removal on days 9 to 12 of menstrual cycle.
§Best result when cumulus is present and Taxol (Bristol-Myers Squibb) was used.
123 Stim MI ? PROH Slow
freeze–rapid thaw
98 Stim GV-MI Yes PROH Slow
freeze–rapid thaw
13 Stim GV PROH Slow
freeze–rapid thaw
98 Unstim
GV-MI
128 Unstim GV Yes PROH Slow
301 Unstim/Stim
GV (PCOD)
78 Unstim
GV-MI
137StimGV Partial/No EG+DMSO/S/F/
§
Yes PROH Slow freeze
freeze–rapid thaw
Yes EG/S/grid
Yes EG/S/grid
HTF/SSS/Cryotoop
Rapid thaw
vitrification
vitrification
vitrification
59 83 62 IVF 58 3 blast
63 68 — — —
23 67 100 ICSI 100 1 live birth
55 59 43 IVF 17 No ET
60 61 — — —
83 68 68 ICSI 90 ET, no
pregnancy
59 64 70 IVF 71 20 embryo
2 blast (7%)
1biochem
79–90 58–85 62–78 ICSI 38–82 1 blast (0.7%)
The concept of short-course FSH stimulation before in vitro
maturation was first introducedbytheseniorauthorin1995while
working in Roger Gosden’s laboratory. However, the effect of a
short (truncated) course of FSH priming (75to 150IU/dayfor 3to
6 days) on immatureoocyte recovery and developmental capacity
is undetermined. Although some researchers [50,53] reported at
least doubling of the number of oocytes reaching metaphase II
oocytes (MII) stage,othersreportedno benefit for suchtreatment
on oocyte recovery and maturation.[54]
On the other hand, the exposure of immature oocytes to
an ovulating dose of hCG 36 hours before retrieval appears to
confer significant benefit on their development. The percentage
of immature oocytes exhibiting germinal vesicle breakdown and
reaching MII stage are increased. In addition, the time required
for in vitro maturation is shortened (24 vs. 48 hours). Synchronization of follicular maturation after hCG assists in timing of
in vitro fertilization by insemination or ICSI.[54,55] Although
denuded oocytes can be matured to MII, their early embryonic development is questionable. The presence of cumulus
cells appears to provide or mediate the action of several hormones/factors needed for IVM.[56]
About 5% to 7% of oocytes retrieved from IVF patients after
ovarian hyperstimulation are immature (germinal vesicle [GV]
stage). A fraction of these oocytes may mature in vitro spontaneously after removal of cumulus cells or by in vitro culture techniques. The developmental capacity of these “leftover” oocytes is
generally low.[49] However, we find this technique most useful
for cancer patients who are undergoing ovarian stimulation for
oocyte or embryo freezing before chemotherapy. In our hands,
IVM with second-day ICSI increases mature oocyte and embryo
yield byat least20% to 25% (unpublished data, Oktay K). Immature oocytes can also be obtained from ovaries removed for gynecologic conditions as well as from ovarian tissue removed for
cryopreservation.[43,57]
Finally, a futuristic use of immature oocytes is for somatic
cell nuclear transfer, and production of customized embryonic
stem cells that are patient compatible for use by the donor of
nuclear material (therapeutic cloning [58]). This concept was
proved in mouse.[59] The erasure of somatic memory is essential for nuclear reprogramming (de-differentiation). This is usually incomplete after nuclear transfer and may lead to severe
developmental abnormalities and poor implantation of cloned
embryos.
IN VITRO MATURATION OF PREANTRAL AND
PRIMORDIAL OOCYTES
Growth and maturation of primordial and preantral follicles is
a major technical challenge for reproductive science. Primordial
follicles (35 μm) are primary oocytes surrounded by one layer
of flattened pregranulosa cells and constitute the ovarian reserve.
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